Methods and compositions for engineered DA neuronal cells

Abstract

New strategies for treating patients suffering from Parkinson's disease and other secondary Parkinson's conditions are disclosed. Disclosed are DA neuronal cells that have been modified in vitro with genetic insertion of GDNF. A GDNF coding sequence is inserted under transcriptional control of a promoter such that after the engineered cell that is to be administered has been mature to a neuronal mature cell type, secretory proteins are produced and uptake by endogenous cells to promote survival of the endogenous neurons. Also disclosed are DA neuronal cells that have been modified in vitro with genetic insertion of GBA and that are SNCA hemizygote-invalid. The GBA coding sequence is inserted under transcriptional control of a traversal promoter and produces secretory proteins after transplantation and uptake immediately by the implant and endogenous cells to promote long term implant integrity.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 579,861, filed August 31, 2023, entitled “Methods and Compositions for Engineered DA Neuronal Cells,” the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list XML, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The attached sequence list is 108 KB in size, named "87874_00116.xml", and was generated on August 13, 2024. Invention Field

[0005] This disclosure generally relates to gene therapy and / or gene editing for the treatment of conditions associated with degeneration of the central nervous system, such as Parkinson's disease. Background Technology

[0006] Gene editing technology

[0007] Since its inception, gene editing technology has proven to be a useful tool in the development of in vitro disease models. The replacement of endogenous genomic sequences with exogenous donor DNA / RNA via homologous recombination (HR) and the correct insertion of exogenous DNA / RNA at designated mammalian chromosomal locations was first developed in the 1980s (Smithies et al., 1984) and subsequently applied to genome modification in mouse embryonic stem cells (PSCs) (Hasty et al., 1991). The discovery of the I-SceI yeast macronuclease (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. This I-SceI yeast macronuclease promotes HR endogenous cellular mechanisms to repair DNA / RNA double-strand breaks (DSBs) in the presence of donor DNA / RNA.

[0008] The advent of zinc finger nuclease (ZFN) technology improved the efficiency of genome editing in mammalian cells (Bibikova et al., 2001), leading to the generation of the first knockout rat (Geurts et al., 2009). Utilizing ZFN-based genome editing in animal and cellular models (Petersen and Niemann, 2015), genetic mutations were corrected 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 known disease-associated mutations were inserted in iPSCs derived from healthy individuals (Verheyen et al., 2018), allowing for direct investigation of specific genomic alterations and disease phenotypes. In addition, ZFNs were applied to generate engineered lines to investigate cell fate determination and improve iPSC differentiation protocols (Hockemeyer et al., 2009), as well as to generate cell type-specific reporter systems for studying disease pathogenesis (Zhang et al., 2016).

[0009] With the advent of transcription activator-like effector nuclease (TALEN), genome editing technology further advanced, demonstrating to be an effective technology for generating animal models (Tesson et al., 2011). Further adoption of TALENs in the study of the nervous system was achieved by introducing disease-causing mutations in control iPSCs (Wen et al., 2014; Lenzi et al., 2015; Akiyama et al., 2019) and / or correcting genetic mutations in patient-derived iPSCs (Maetzel et al., 2014; Wen et al., 2014; Li H. L. et al., 2015; Tanaka et al., 2018; Akiyama et al., 2019)), leading to greater confidence in the fundamental mechanisms of disease and therapeutic method development. In addition, reporter lines for stem cell-based research were developed using TALEN technology (Cerbini et al., 2015; Pei et al., 2015).

[0010] Rapidly following the development of TALEN technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas13) system (Gasiunas et al., 2012; Jinek et al., 2012) demonstrated revolutionary potential for engineering the mammalian cell genome in culture (Cong et al., 2013; Mali et al., 2013) and animal models (Wang H. et al., 2013). Like ZFNs or TALENs, CRISPR-Cas13 uses a different RNA cleavage and binding module. However, the CRISPR-Cas13 system uses its own native endonuclease and relies on CRISPR RNA (crRNA) and trans-activating RNA (transRNA) to specifically bind to target RNA sequences and activate Cas13. Thus, the lengthy and complex process of engineering a nuclease was rapidly overcome by the plasticity and simplicity of generating different CRISPR-based approaches that only required the design of a specific target-matching RNA. The extraordinary efficacy of CRISPR-Cas13 and its greater versatility in generating a wide range of substitutions, duplications, deletions, inversions, and many other complex alterations up to chromosomal rearrangements has revolutionized the field of genome editing. However, there are several limitations that need further improvements. By engineering Cas13 proteins (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) and modifications of the design and structure of the guide RNA (Jinek et al., 2012; Hsu et al., 2013; Cui et al., 2018; Filippova et al., 2019; Moon et al., 2019), as well as the discovery and application of Cas proteins with different and specific gene editing properties (Zetsch et al., 2015; Abudayyeh et al., 2016; Burstein et al., 2017), improvements in efficiency and reduction of off-target effects have been achieved. CRISPR-based technologies have further evolved to allow transcription repression (CRISPR interference, CRISPRi) or activation (CRISPR activation, CRISPRa). This CRISPR-based transcriptional modulation is achieved by fusing repressor or activator transcription domains to catalytically inactive Cas13 (dCas13) and guide RNAs to specific genes’ promoters or regulatory regions (Gilbert et al., 2013).

[0011] CRISPR-based engineering technologies have enabled researchers to dissect the function of specific genetic elements or correct pathogenic mutations. In parallel, CRISPR tools are now being implemented to perform active control and modulation of desired messenger RNA (mRNA). This allows interrogation of transcriptomic kinetics and establishment of causal links between observed transcriptional changes and cellular phenotypes. Previously, RNA interference (RNAi) technologies enabled the inhibition of desired transcripts using microRNAs (miRNAs), but this came with significant off-target effects due to cross-reactivity with targets of limited sequence similarity and erroneous targeting effects associated with endogenous miRNAs (Flynt and Lai, 2008). Studies on Cas proteins capable of targeting RNA led to the development of an RNA-guided and RNA-targeting enzyme (CasRx) (Konermann et al., 2018) that showed improved efficiency in knocking down endogenous mRNA levels compared to RNAi technologies, allowing for facile manipulation of alternative splicing in human cells. Furthermore, Konermann and colleagues successfully applied Cas-Rx editing to a patient-derived FTD cortical neuron model to modulate the balance of tau isoforms. Certain forms of FTD associated with parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies are caused by mutations in the intron after exon 10 of MAPT (Boeve and Hutton, 2008). These variants disrupt the intronic splice site and increase the expression of the 4R tau isoform, which contains more microtubule-binding domains (Kar et al., 2005), inducing pathological changes and driving the progression of neurodegeneration (Schoch et al., 2016). CasRx-mediated exon exclusion reduced 4R tau expression to levels similar to unaffected control neurons, suggesting that this technology can be used for transcriptional modulation in in vitro models. Interestingly, the small size of CasRx is amenable to packaging in adeno-associated virus (AAV) for delivery into post-mitotic neurons, encouraging future clinical applications in treating neurological diseases and can be paired with arrays encoding multiple guide RNAs for multiplexing. Thus, CasRx technology paves the way for transcriptomic engineering and RNA-targeting therapeutic applications.

[0012] Differentiation into lineage-specific cell populations

[0013] Methods are needed to generate DA neuronal cells from pluripotent cells, as such cells can be used both therapeutically and in disease models, e.g., to identify new therapeutics for treating Parkinson’s disease, as well as other and secondary Parkinsonian conditions, including but not limited to idiopathic Parkinson’s disease, vascular parkinsonism, drug-induced parkinsonism, and non-idiopathic Parkinsonian conditions, including but not limited to Parkin and other familial and genetic diseases.

[0014] Various efforts have been made to generate midbrain DA neurons from pluripotent cells. For example, methods to generate midbrain DA neurons from pluripotent cells generally require the use of both the BMP signaling inhibitor LDN-193189 (inhibits ALK 1 / 2 / 3 / 6, blocks SMAD 1 / 5 / 8) and the TGF-b signaling inhibitor SB-431542 (inhibits ALK 4 / 5 / 7, blocks SMAD 2 / 3), as described, e.g., in U.S. Patent No. 10,280,398, which is hereby incorporated by reference in its entirety. As these methods utilize a combination of two Small Mothers Against Decapetaplegic (SMAD) signaling inhibitors, these methods are generally referred to as “dual SMAD inhibition” or “dual SMADi.”

[0015] A method of preparing DA neurons using dual SMAD inhibition includes differentiating pluripotent stem cells, including exposing a plurality of pluripotent stem cells to at least one TGFp / Activin-Nodal signaling inhibitor, at least one bone morphogenetic protein (BMP) signaling inhibitor, at least two Sonic hedgehog (SHH) signaling activators (such as purmorphamine and SHH C25II), and at least one glycogen synthase kinase 3 beta (GSK3B) signaling inhibitor that activates wingless (Wnt) signaling, wherein the cells are exposed to the at least one TGFp / Activin-Nodal signaling inhibitor and the at least one BMP signaling inhibitor beginning on day 0, wherein the cells are exposed to the at least one GSK3B signaling inhibitor from the third (3rd) day to the eleventh (11th) day of initial exposure of the cells to the at least one TGFp / Activin-Nodal signaling inhibitor and the at least one BMP signaling inhibitor, in an amount effective for producing a cell population comprising at least about 10% differentiated cells that express both forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A). Another method of preparing DA neurons using dual SMAD technology is disclosed in U.S. Patent Nos. 10,858,625 and 10,273,452, which are incorporated by reference in their entirety. A method of obtaining an enriched population of midbrain dopaminergic (DA) neurons is described in U.S. Patent No. 10,828,335, which is incorporated by reference in its entirety. A method of preparing pluripotent stem cells for neural differentiation is described in U.S. Patent No. 9,487,752, which is incorporated by reference in its entirety.

[0016] Others use single-SMAD inhibition (single-SMADi) to generate midbrain DA neurons from pluripotent cells. See, e.g., U.S. Patent No. 10,590,383, which is incorporated by reference herein in its entirety. Generally, the method includes culturing human pluripotent cells in the presence of: (a) a single inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, (b) at least one Sonic hedgehog (SHH) signaling activator, and (c) at least one wingless (Wnt) signaling activator; and culturing the cells in the presence of the modulators for a time sufficient to provide a cell composition comprising FOXA2+ / LMX1+ cells; wherein the culturing does not include culturing the human pluripotent cells in the presence of a second Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor.

[0017] GBA deficiency promotes SNCA / alpha-synuclein accumulation

[0018] Loss-of-function mutations in the gene encoding GBA (glucocerebrosidase, beta, acid) (an enzyme deficient in Gaucher disease, a lysosomal storage disorder) increase the risk of Parkinson’s disease (PD), which is characterized by misprocessing of SNCA / alpha-synuclein. It has been discovered that loss of GBA function leads to increased SNCA levels via inhibition of the autophagic 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 / alpha-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 can contribute to SNCA accumulation by inhibiting autophagy via PPP2A inactivation, thereby providing a mechanistic basis for the increased risk of PD associated with GBA deficiency.

[0019] There is a great need in the art for new compositions and methods for treating and preventing degeneration of the central nervous system. SUMMARY

[0020] DA neuron cells with delayed exogenous GDNF expression

[0021] In cell transplantation therapy, a major challenge is to avoid cells with low survival, engraftment, proliferation, migration, innervation, differentiation, or function. This is more likely to occur when the engineered gene is partially expressed or not fully expressed, expressed at the wrong time, or misregulated during expression.

[0022] To avoid these deficiencies, it is desirable to include transcriptional regulation such that the gene is not expressed until after the implant has been established, i.e., after the implant has incorporated into host tissue. In some embodiments, the exogenous gene under the control of an endogenous promoter is not expressed until the transplanted cell (DA neuron cell) has differentiated in vivo to a more mature cell type, such as an immature neuron, mature neuron, or neuron. Delayed expression of a knock-in gene such as GDNF is expected to increase the therapeutic potential of the transplanted engineered DA neuron cell by improving the function of the neuron mature cell type that the transplanted engineered DA neuron cell differentiates to in vivo. Delayed expression of a knock-in gene such as GDNF is expected to increase the therapeutic potential of the transplanted engineered DA neuron cell by improving the function of the neuron mature cell type (i.e., endogenous neuron mature cell type) found in the intracranial, intranuclear environment near the transplanted engineered DA neuron cell. By using gene targeting insertion with sequence-specific endonuclease reagents to introduce exogenous gene sequences to be expressed or co-expressed after the transplanted cell has differentiated in vivo to a neuron mature cell type, such that their coding sequences are transcribed under the control of an endogenous promoter present at a selected locus expressed in the neuron mature cell type. Alternatively, loci that are not expressed during neural cell differentiation can be used as “safe harbor loci” for integration of expression cassettes without any adverse consequences to the engineered pluripotent cell differentiating in vitro to an engineered DA neuron cell or the transplanted DA neuron cell differentiating in vivo to a neuron mature cell type. In some embodiments, the selected site for exogenous gene insertion is a safe harbor locus, a highly expressed locus, a transiently expressed locus, or a locus for interruption. In some embodiments, the safe harbor locus is AAVS1, CCR5, hROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or a locus that meets the genomic safe harbor criteria as defined herein. In some embodiments, the gene locus for interruption comprises GDNF, GBA PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA. In some embodiments, the safe harbor locus is selected from the list in Table 1.

[0023] These cell engineering strategies generally tend to enhance the therapeutic potential of the transplanted engineered DA neuronal cells, particularly by increasing their viability, engraftment, proliferation, migration, innervation, and / or differentiation, or by increasing the function of the neuronal mature cell types into which the transplanted engineered DA neuronal cells differentiate in vivo and / or increasing the survival of endogenous neurons. This strategy can be performed on patient-derived cells as part of an autologous therapeutic strategy, as well as on donor-derived cells as part of an allogeneic therapeutic strategy.

[0024] DA neuronal cells expressing exogenous GBA immediately and SNCA hemizygously null

[0025] Expression of a knock-in gene such as GBA once the DA neuronal cells are transplanted is expected to increase the therapeutic potential of the transplanted engineered DA neuronal cells by improving long-term graft integrity. The exogenous genetic sequence is expressed or co-expressed post-transplantation. In some embodiments, the exogenous genetic sequence is transcribed under the control of an endogenous ubiquitous promoter. Alternatively, the locus of a gene previously identified as lacking endogenous expression during DA neuronal cell transplantation can be used as a "safe harbor locus" for integration of an expression cassette under the control of a ubiquitous promoter.

[0026] Engineered pluripotent cell populations

[0027] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous knock-in polynucleotide sequence encoding a wild-type Parkinson 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), vesicular monoamine transport protein (VMAT2), glucocerebrosidase (GBA), and / or glial cell line-derived neurotrophic factor (GDNF) gene. In some embodiments, the knock-in polynucleotide sequence is under the control of a GDNF promoter (SEQ ID NOs: 21-30). In some embodiments, the knock-in polynucleotide sequence is under the control of a GBA promoter (SEQ ID NOs: 31-40 or 43).

[0028] Engineered DA neuronal cell populations

[0029] Disclosed are engineered DA neuronal cell populations, wherein the engineered DA neuronal cells comprise an exogenously knocked-in PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / - and / or GDNF gene. In some embodiments, the knocked-in polynucleotide sequence is under the control of a GDNF promoter (SEQ ID NOs: 21-30). In some embodiments, the knocked-in polynucleotide sequence is under the control of a GBA promoter (SEQ ID NOs: 31-40 or 43).

[0030] Gene editing system

[0031] The engineered pluripotent cells and / or engineered DA neuronal cells discussed above can be made by any known gene editing system known to one of skill in the art. Thus, the engineered pluripotent cells and / or engineered DA neuronal cell populations discussed above can comprise a nuclease (Cas protein, TALE nuclease, or zinc finger nuclease), a repair template comprising a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygously null SCNA, and / or GDNF gene, or functional fragment or variant thereof.

[0032] The engineered pluripotent cells and / or engineered DA neuronal cells discussed above can be made by any known gene editing system known to one of skill in the art. Thus, the engineered pluripotent cells and / or engineered DA neuronal cell populations discussed above can comprise a Cas protein, a guide RNA, a repair template comprising a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygously null SCNA, and / or GDNF gene, or functional fragment or variant thereof. Thus, the engineered pluripotent cells and / or engineered DA neuronal cell populations discussed above can comprise a nuclease (Cas protein, TALE nuclease, or zinc finger nuclease), a repair template comprising a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / - and / or GDNF gene, or functional fragment or variant thereof, in combination with editing (or disruption) of the SNCA and / or MAPT gene, to render the cell population hemizygously null for SNCA and / or MAPT. In some embodiments, the repair template does not comprise SCNA.

[0033] Recombinant gene vector

[0034] Various embodiments of recombinant genetic vectors comprising a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene or functional fragment or variant thereof, and methods related to the recombinant genetic vectors are disclosed. Various embodiments of recombinant genetic vectors comprising 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 a complement or RNA equivalent thereof, and methods related to the recombinant genetic vectors are disclosed.

[0035] Methods

[0036] Contacting pluripotent cells with a gene editing system in vitro

[0037] In some embodiments, methods of making an engineered DA neuronal cell are disclosed, comprising contacting a pluripotent cell 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, hemizygous SNCA, hemizygous MAPT, and / or GDNF gene or 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 a complement or RNA equivalent thereof.

[0038] In some embodiments, methods of making engineered DA neuronal cells are disclosed, comprising contacting a pluripotent cell with a gene editing system comprising a Cas protein or a polynucleotide encoding a Cas protein; a guide RNA (gRNA); and a repair template comprising 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 selected from the group comprising or consisting of SEQ ID NOs: 1-10. In some embodiments, the gene editing system is capable of enhancing the viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cells compared to wild-type neuronal cells. In some embodiments, the administered engineered DA neuronal cells differentiate in vivo into neuronal mature cell types with enhanced function as they are derived from the administered engineered DA neuronal cells. In some embodiments, the administered engineered DA neuronal cells are administered in vivo and have an effect on endogenous neuronal mature cell types.

[0039] Contacting a neural cell with a gene editing system ex vivo or in vivo

[0040] In some embodiments, methods of contacting a neuron with a gene editing system are disclosed, the 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 selected from the group comprising or consisting of SEQ ID NOs: 1-10.

[0041] In some embodiments, methods of contacting a neuron with a gene editing system are disclosed; the gene editing system comprises a Cas protein or a polynucleotide encoding a Cas protein, a guide RNA (gRNA); 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 selected from the group comprising or consisting of SEQ ID NOs: 1-10. In some embodiments, the gene editing system is capable of enhancing survival, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cell compared to a wild-type neuronal cell. In some embodiments, the neuron is in vivo in a patient when it is contacted with the engineered DA neuronal cell. In some embodiments, the neuron is contacted with the gene editing system ex vivo and then transplanted back into the patient in vivo after the contacting step.

[0042] Contacting an endogenous wild-type neural cell with an engineered DA neuronal cell in vivo

[0043] In some embodiments, methods of contacting a neuron with an engineered DA neuronal cell are disclosed, comprising contacting a pluripotent cell 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, 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 selected from the group comprising or consisting of SEQ ID NOs: 1-10.

[0044] In some embodiments, methods of contacting a neuron with an engineered DA neuronal cell are disclosed, comprising contacting a pluripotent cell with a gene editing system; the gene editing system comprises a Cas protein or a polynucleotide encoding a Cas protein, a guide RNA (gRNA); and a repair template comprising 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 selected from the group comprising or consisting of SEQ ID NOs: 1-10. In some embodiments, the engineered DA neuronal cell is capable of enhancing viability, engraftment, proliferation, migration, innervation, differentiation, long-term implant integrity, survival of an endogenous wild-type neuron, or a function of an endogenous wild-type neuronal cell compared to a non-contacted wild-type neuronal cell. In some embodiments, the endogenous wild-type neuron is in a patient when it is contacted with the engineered DA neuronal cell. In some embodiments, the endogenous wild-type neuron is contacted with the engineered DA neuronal cell ex vivo and then transplanted back into the patient in vivo after the contacting step.

[0045] Transplantation

[0046] The engineered DA neuronal cell, recombinant gene vector, or gene editing system can be administered in various ways. In some embodiments, the administering step comprises systemic, parenteral, intravenous, brain, cerebrospinal, intrathecal, intracisternal, intranucleus lentiformis, intrahippocampal, intrastriatal, or intracerebroventricular administration. In some embodiments, the administering step comprises intravenous, brain, cerebrospinal, intrathecal, intracisternal, intranucleus lentiformis, intrahippocampal, intrastriatal, or intracerebroventricular injection. In some embodiments, the administering step comprises intrathecal injection with Threndelenburg tilting. In some embodiments, the administering step comprises direct injection into the substantia nigra compacta of the brain. In some embodiments, the administering step comprises introducing the engineered DA neuronal cell, recombinant gene vector, or gene editing system into the brain or cerebrospinal fluid (CSF) of the subject.

[0047] In some embodiments, 1 x 10 9 - 1 x 10 14 recombinant gene vector genomes per kilogram of subject body weight (vg / kg) are administered to the brain of the subject. In some embodiments, 1 x 10 9 - 1 x 10 141 x 107recombinant gene vector genomes per kilogram of subject body weight (vg / kg). In some embodiments, 1 x 105 9 1 x 107recombinant gene vector genomes per kilogram of subject body weight (vg / kg). In some embodiments, 1 x 105 14 1 x 107recombinant gene vector genomes per kilogram of subject body weight (vg / kg). In some embodiments, 1 x 105 7 1 x 107recombinant gene vector genomes per kilogram of subject body weight (vg / kg). In some embodiments, 1 x 105 9 1 x 107recombinant gene vector genomes per kilogram of subject body weight (vg / kg).

[0048] Treatment outcomes

[0049] The methods of the present disclosure (administering engineered DA neuronal cells, recombinant gene vectors, or gene editing systems) can have various effects. In some embodiments, the administered engineered DA neuronal cells have increased viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cells. In some embodiments, the administered engineered DA neuronal cells do not have altered effects. In some embodiments, the administered engineered DA neuronal cells do not have increased viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cells. In some embodiments, only cells differentiated from the administered engineered DA neuronal cells (e.g., neuronal mature cell types) have altered effects. In some embodiments, the administered engineered DA neuronal cells cause endogenous neuronal mature cell types to have increased viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cells.

[0050] In some embodiments, the administered engineered DA neuronal cells with an engineered GBA gene have increased cell viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cells. In some embodiments, the administered engineered DA neuronal cells with an engineered GBA gene and a disrupted SNCA gene have increased cell viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cells. In some embodiments, the administered engineered DA neuronal cells with an engineered GDNF gene have increased cell viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cells. In some embodiments, the administration of the engineered DA neuronal cells treats or inhibits the onset of Parkinson’s disease and secondary Parkinsonian conditions in the subject. In some embodiments, the administration of the engineered DA neuronal cells treats or inhibits the onset of bradykinesia. In some embodiments, the administration of the engineered DA neuronal cells treats or inhibits the onset of mental retardation. In some embodiments, the administration of the engineered DA neuronal cells treats or inhibits the onset of neurochemical-related conditions or decline associated with impairment of dopamine, acetylcholine, serotonin, and / or norepinephrine signaling.

[0051] Sequence

[0052] In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF gene (or vector) comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-10, respectively.

[0053] In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11-20, respectively.

[0054] In some embodiments, the gene is a GDNF gene, and the wild-type GDNF protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10.

[0055] In some embodiments, the gene is a GBA gene, and the wild-type GBA protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7.

[0056] In some embodiments, the polynucleotide comprises a sequence that is at least 70%, 75%, 80%, 85%, 95%, or 99% identical to the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, or GDNF polynucleotide sequence set forth 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 to 20 CpG islands.

[0057] Sequence ID

[0058] SEQ ID No: 1: > Homo sapiens PARK2 ENSG00000185345.24

[0059]

[0060]

[0061] SEQ ID No: 2: > Homo sapiens PINK1 ENSG00000158828.8

[0062]

[0063]

[0064] SEQ ID No: 3: > Homo sapiens LRRK2 ENSG00000188906.17

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] SEQ ID No: 4: > Homo sapiens c-Rel ENSG00000162924.16

[0071]

[0072]

[0073] SEQ ID No: 5: > Homo sapiens ATG7 ENSG00000197548.13

[0074]

[0075]

[0076] SEQ ID No: 6: > Homo sapiens VMAT2 ENSG00000165646.14

[0077]

[0078]

[0079] SEQ ID No: 7: > Homo sapiens GBA ENSG00000177628.16

[0080]

[0081]

[0082] SEQ ID No: 8: > Homo sapiens SNCA ENSG00000145335.17

[0083]

[0084]

[0085] SEQ ID No: 9: > Homo sapiens MAPT ENSG00000186868.18

[0086]

[0087]

[0088] SEQ ID No: 10: > Homo sapiens GDNF ENSG00000168621.15

[0089]

[0090]

[0091] SEQ ID No: 11: > Homo sapiens PARK2 ENSG00000185345.24

[0092]

[0093] SEQ ID No: 12: > Homo sapiens PINK1 ENSG00000158828.8

[0094]

[0095] SEQ ID No: 13: > Homo sapiens LRRK2 ENSG00000188906.17

[0096]

[0097]

[0098]

[0099] SEQ ID No: 14: > Homo sapiens c-Rel ENSG00000162924.16

[0100]

[0101] SEQ ID No: 15: > Homo sapiens ATG7 ENSG00000197548.13

[0102]

[0103]

[0104] SEQ ID No: 16: > Homo sapiens VMAT2 ENSG00000165646.14

[0105]

[0106] SEQ ID No: 17: > Homo sapiens GBA ENSG00000177628.16

[0107]

[0108] SEQ ID No: 18: > Homo sapiens SNCA ENSG00000145335.17

[0109]

[0110] SEQ ID No: 19: > Homo sapiens MAPT ENSG00000186868.18

[0111]

[0112] SEQ ID No: 20: > Homo sapiens GDNF ENSG00000168621.15

[0113] BRIEF DESCRIPTION OF DRAWINGS

[0114] Figure 1 Different neural lineage cells and their markers are depicted.

[0115] Figure 2 A research strategy to identify genome safe harbor (GSH) sites suitable for iPSC gene editing and expression in dopaminergic neurons is depicted.

[0116] Figure 3 Is an illustration of a stepwise approach to prioritize GSH sites for therapeutic gene insertion. DETAILED DESCRIPTION

[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of gene therapy, biochemistry, genetics, and molecular biology.

[0118] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0119] The practice of gene editing disclosed herein can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are explained fully in the literature. See, e.g., 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 (M. J. Gait ed., 1984); Mullis et al. U.S. Patent Number 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon eds., Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos, ed., 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 I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0120] Generally, disclosed herein are compositions comprising engineered cells. The engineered cells can be administered to a subject in a therapeutically effective amount. Administration of the engineered cells can result in a therapeutic outcome in the subject, wherein the therapeutic outcome is modulated by an exogenous gene added to the cell.

[0121] Definitions

[0122] Amino acid residue

[0123] Amino acid residues in a polypeptide sequence are designated herein according to the one-letter code, wherein, for example, Q designates Gin or glutamine residue, R designates Arg or arginine residue, and D designates Asp or aspartic acid residue.

[0124] Amino acid substitution

[0125] Amino acid substitution means the replacement of one amino acid residue with another, for example, the replacement of an arginine residue with a glutamine residue in a peptide sequence is an amino acid substitution.

[0126] Cleavage

[0127] The term "cleavage" refers to the breakage 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 cleavage and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two distinct single-strand cleavage events. Double-stranded DNA, RNA, or DNA RNA hybrid cleavage can result in the production of either blunt or staggered ends.

[0128] DA neuron cell

[0129] DA neuronal cells can be differentiated in vitro from pluripotent cells by a variety of methods known in the art, for example by single-SMAD or dual-SMAD technology as discussed herein.

[0130] DNA target

[0131] “DNA target,” “DNA target sequence,” “target DNA sequence,” “nucleic acid target sequence,” “target sequence,” or “processing site” refers to a polynucleotide sequence that is targetable and processable by a rare-cutting endonuclease. These terms refer to a specific DNA location in a cell, preferably a genomic location, but also to a portion of genetic material that can exist independently of the bulk of genetic material, as non-limiting examples, such as a plasmid, episome, virus, transposon, or in an organelle, such as a mitochondrion. Non-limiting examples of RNA-guided target sequences are those genomic sequences that are hybridizable to a guide RNA that directs an RNA-guided endonuclease to the desired locus.

[0132] Dopaminergic neuron

[0133] Dopaminergic neuron is a cell that expresses TH, DAT, FOXA2, GIRK2, Nurr1, and LMX1B.

[0134] Engineered DA neuronal cell

[0135] “Engineered DA neuronal cell” means a population of DA neuronal cells comprising an exogenous knock-in of a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF gene.

[0136] Enhanced therapeutic activity

[0137] “Enhanced therapeutic activity” means that a DA neuronal cell or population of cells engineered as described herein becomes more viable, or has improved engraftment, proliferation, migration, innervation, and / or differentiation than a non-engineered cell or population of cells.

[0138] Endonuclease

[0139] The term "endonuclease" refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. Endonucleases do not cleave DNA or RNA molecules irrespective of the sequence of said DNA or RNA molecule, but rather recognize and cleave the DNA or RNA molecule at specific polynucleotide sequences, further referred to as "target sequences" or "target sites". Endonucleases can be classified as rare-cutting endonucleases when the polynucleotide recognition site typically has a length of more than 10 base pairs (bp), more preferably 14-55 bp. Rare-cutting endonucleases significantly increase homologous recombination by inducing DNA double strand breaks (DSBs) at defined loci, thereby allowing gene repair or gene insertion therapy (Pingoud, A. and G. H. Silva (2007). PreSNCAion genome surgery. Nat. Biotechnol. 25(7): 743-4.).

[0140] Exogenous sequence

[0141] An "exogenous sequence" refers to any nucleotide or nucleic acid sequence that is not originally present at the selected locus. The sequence can be homologous to or a copy of a genomic sequence, or an exogenous sequence introduced into the cell. In contrast, an "endogenous sequence" means a cellular genomic sequence originally present at the locus. Preferably, the exogenous sequence encodes a polypeptide whose expression confers a therapeutic advantage relative to a sister cell that has not integrated this exogenous sequence at the locus. Endogenous sequences that are genetically edited to express different polypeptides by insertion of nucleotides or polynucleotides according to the disclosed methods are broadly referred to as exogenous coding sequences

[0142] The methods disclosed herein can be combined with other methods involving physical genetic transformation, such as viral transduction or transfection using nanoparticles, and also with other gene inactivation and / or gene insertion.

[0143] GBA gene

[0144] The term "GBA gene" encompasses the GBA gene and functional fragments and variants thereof.

[0145] GDNF gene

[0146] The term "GDNF gene" encompasses the GDNF gene and functional fragments and variants thereof.

[0147] GABAergic neurons

[0148] GABAergic neurons are cells expressing GAT1, GABAB receptor 1, GABAB receptor 2, GAD65 and GAD67.

[0149] gRNA molecule

[0150] A gRNA molecule can be positioned at, comprise, consist of, or consist essentially of a nucleic acid sequence that is fully or partially complementary to a target domain (e.g., a gene or a portion of a gene). In certain embodiments, a single molecule or chimeric gRNA comprises, preferably from 5' to 3': a targeting domain that is complementary to a target domain in a nucleotide in a cell (e.g., a chromosome); a first complementarity domain; a linker domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and, optionally, a tail domain.

[0151] Gene editing system

[0152] A gene editing system, or genome editing system, is a group of technologies that give scientists the ability to change DNA in living organisms. These technologies allow the addition, removal, or alteration of genetic material at specific locations in the genome.

[0153] Genomic safe harbor

[0154] A genomic safe harbor (GSH) is a site in the genome that is able to accommodate integration of new genetic material in a way that ensures that the newly inserted genetic element: (i) functions predictably, and (ii) does not cause changes to the host genome that pose a risk to the host cell or organism. Table 1 below is a list of genomic safe harbor loci that can be used, as described herein. Table 1 shows genomic coordinates in the GRCh38 / hg38 human genome assembly.

[0155] Table 1

[0156] chromosome start end chr1 214186905 214187961

[0157]

[0158]

[0159]

[0160]

[0161] Gene targeting integration

[0162] “Gene targeting integration” refers to any known site-specific method that allows for the insertion, replacement, or correction of genomic sequences in living cells. In some embodiments, gene targeting integration involves homologous gene recombination at the locus of a targeted gene to result in the insertion or replacement of at least one exogenous nucleotide, preferably several nucleotides of sequence (i.e., a polynucleotide), more preferably a coding sequence.

[0163] Glutamatergic neurons

[0164] Glutamatergic neurons are cells expressing VGLUT1, VGLUT2, NMDAR1, NMDAR2B, glutaminase, and glutamine synthetase.

[0165] Identity

[0166] “Identity” refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between a nucleic acid or amino acid sequence is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, 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, under default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to a particular polypeptide described herein and preferably exhibiting essentially the same function, as well as polynucleotides encoding such polypeptides, are contemplated.

[0167] Immature neurons

[0168] Immature neurons are cells expressing Doublecortin, NeuroDl, TBR1, beta III tubulin, and Stathmin 1. Immature neurons can differentiate into mature neurons.

[0169] Improved therapeutic potential

[0170] “Improved therapeutic potential” refers to the acquisition by engineered DA neuronal cells of at least one favorable property for their use in cell therapy as compared to their sister non-engineered DA neuronal cells. The therapeutic property sought can be any measurable property as mentioned in the relevant scientific literature and includes, but is not limited to, the viability, engraftment, proliferation, migration, innervation, and / or differentiation of the administered engineered DA neuronal cells.

[0171] The improved therapeutic potential can more specifically reflect an increased resistance of the DA neuronal cells to drugs, an increase in their in vitro or in vivo persistence, or a safer / more convenient handling during the preparation of the therapeutic composition and during the treatment.

[0172] Generally, the molecule that improves the therapeutic potential is a polypeptide, but it can also be a nucleic acid capable of directing or inhibiting the expression of other genes, such as an interfering RNA or a guide RNA. The polypeptide can act directly or indirectly, for example as a signal transducer or a transcription regulator.

[0173] locus

[0174] As used herein, the term "locus" is the specific physical location into which a DNA sequence (e.g., a gene) enters the genome. The term "locus" can refer to the specific physical location on a chromosome or on the genome sequence of an infectious source of a rare-cutting endonuclease target sequence. Such loci can comprise a target sequence recognized and / or cleaved by a sequence-specific endonuclease. It will be appreciated that a locus of interest can refer not only to a nucleic acid sequence present in the bulk of the genetic material of a cell (i.e., in a chromosome), but also to a portion of the genetic material that can exist independently of the bulk of the genetic material, such as, by way of non-limiting example, a plasmid, an episome, a virus, a transposon, or in an organelle, such as a mitochondrion.

[0175] mature neuron

[0176] A mature neuron is a cell that expresses NeuN, MAP2, 160 kDa intermediate neurofilament, heavy neurofilament, synapsin, and PSD95. A mature neuron can differentiate into a neuron.

[0177] modified DA neuron cell

[0178] A "modified DA neuron cell" or "engineered DA neuron cell" means a DA neuron cell that has been genetically modified itself or is derived from a genetically modified pluripotent cell.

[0179] modified pluripotent cell

[0180] A "modified pluripotent cell" or "engineered pluripotent cell" refers to a pluripotent cell that has been genetically modified.

[0181] mutation

[0182] "Mutation" means a substitution, deletion, insertion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more nucleotides / amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. The mutation can affect the coding sequence of a gene or its regulatory sequences. It can also affect the structure of the genomic sequence or the structure / stability of the encoded mRNA.

[0183] neuron

[0184] Glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, cholinergic neurons are collectively referred to as "neurons".

[0185] nucleotide

[0186] Nucleotides are designated as follows: the one-letter code is used to designate the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For degenerate nucleotides, r denotes g or a (purine nucleotides), k denotes g or t, s denotes g or c, w denotes a or t, m denotes a or c, y denotes t or c (pyrimidine nucleotides), d denotes g, a or t, v denotes g, a or c, b denotes g, t or c, h denotes a, t or c, n denotes g, a, t or c.

[0187] Nucleic acid

[0188] As used herein, “nucleic acid” or “polynucleotide” refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by any of ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can comprise monomers of naturally occurring nucleotides (e.g., DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or pyrimidine or purine base moieties. Sugar modifications include, for example, the replacement of one or more hydroxyls with halogens, alkyls, amines, and azides, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures such as azase sugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic analogs of these bases. The nucleic acid monomers can be joined by phosphodiester bonds or analogs of such linkages. Nucleic acids can be single-stranded or double-stranded.

[0189] Nuclease agent

[0190] “Nuclease agent” refers to a nucleic acid molecule that, by itself or as a subunit of a complex (e.g., a guide RNA / Cas protein), contributes to a nuclease catalytic reaction (preferably an endonuclease reaction) in a target cell, preferably resulting in cleavage of a target of a nucleic acid sequence.

[0191] Nuclease agents used herein are generally “sequence-specific agents,” meaning that they can induce DNA cleavage at a predetermined locus in a cell, referred to as a “targeted gene.” The nucleic acid sequence recognized by a sequence-specific agent is referred to as a “target sequence.” The target sequence is typically chosen to be rare or unique in the genome of the cell, and more broadly in the human genome, as can be determined using software and data available from human genome databases such as http: / / www.ensembl.org / index.html

[0192] Neuronal mature cell type

[0193] ​Neuronal mature cell types refer to immature neurons, mature neurons, or neurons in general.

[0194] Patient

[0195] As used herein, the term “subject” or “patient” includes all members of the animal kingdom, including non-human primates and humans.

[0196] Promoters for GDNF knock-in include SEQ ID NOs: 21-30 (these sequence lists are “synthetic constructs”)

[0197] Monoamine oxidase B (MAOB) SEQ ID NO: 21:

[0198]

[0199]

[0200] GTP cyclohydrolase 1 (GCH1) SEQ ID NO: 22

[0201]

[0202] Nuclear receptor subfamily 4 group A member 2 (NR4A2) SEQ ID NO: 23

[0203]

[0204] Mouse tyrosine hydroxylase (TH) SEQ ID NO: 24

[0205]

[0206]

[0207]

[0208] Solute carrier family 6 member 3 (SLC6A3) SEQ ID NO: 25

[0209]

[0210]

[0211] Synapsin 1 (SYN1) SEQ ID NO: 26

[0212]

[0213] Mouse alpha-Ca2+-calmodulin-dependent kinase II (Camk2a) SEQ ID NO: 27

[0214]

[0215]

[0216] Neurofilament medium (NEFM) mini-promoter SEQ ID NO: 28

[0217]

[0218]

[0219] Neurofilament light (NEFL) mini-promoter SEQ ID NO: 29

[0220]

[0221]

[0222] Neurofilament heavy (NEFH) promoter SEQ ID NO: 30

[0223]

[0224]

[0225] Promoters for GBA knock-in include the following SEQ ID NOS: 31-40 and 43 (these sequence listings are “synthetic constructs”):

[0226] Mouse Thy-1 cell surface antigen (mTHY1) SEQ ID NO: 31

[0227]

[0228]

[0229] Rat neuron-specific enolase (rNSE) promoter SEQ ID NO: 32

[0230]

[0231]

[0232] Dopa decarboxylase (DDC) promoter SEQ ID NO: 33

[0233]

[0234] Catechol-O-methyltransferase (COMT) promoter SEQ ID NO: 34

[0235]

[0236] Glucosylceramidase beta 1 promoter 1 (GBA P1) SEQ ID NO: 35

[0237]

[0238]

[0239] Glucosylceramidase beta 1 promoter 2 (GBA P2) SEQ ID NO: 36

[0240]

[0241] Human eukaryotic translation elongation factor 1 alpha 1 short form (EFS) promoter SEQ ID NO: 37

[0242]

[0243] Human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter SEQ ID NO: 38

[0244]

[0245]

[0246] Chicken beta actin (CBA) promoter SEQ ID NO: 39

[0247]

[0248] Cytomegalovirus (CMV) promoter SEQ ID NO: 40

[0249]

[0250] Human phosphoglycerate kinase 1 (hPGK) promoter SEQ ID NO: 43

[0251]

[0252]

[0253] Pluripotent cell

[0254] “Pluripotent cell” refers to a cell that is capable of differentiating into cells of all three germ layers. Pluripotent cells include stem cells, such as umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (IPSs).

[0255] Rare-cutting endonuclease

[0256] “Rare-cutting endonucleases” are sequence-specific endonuclease reagents of choice because their recognition sequences typically range from 10 to 50 contiguous base pairs, preferably from 12 to 30 bp, more preferably from 14 to 20 bp.

[0257] Serotonergic neurons

[0258] Serotonergic neurons are cells that express TPH, SERT, and Petl.

[0259] SNCA-modified DA neuron cells

[0260] As used herein, the term “SNCA-modified DA neuron cell” refers to a DA neuron cell in which SNCA activity is inhibited by any one of a variety of strategies, alone or in combination. For example, SNCA-modified DA neuron cells include, but are not limited to, SNCA “knockout” DA neuron cells in which the SNCA gene has been genetically deleted or modified, e.g., by gene editing, i.e., SNCA - / - In some embodiments, only a single allele of the SNCA gene is affected by gene editing, i.e., the cell is a SNCA + / - “knockdown” DA neuron cells in which expression of SNCA protein has been reduced by use of gene silencing strategies (e.g., use of siRNA or RNAi) or expression of dominant negative SNCA sequence variants or dominant negative SNCA fragments; or, the SNCA-modified DA neuron cell is a DA neuron cell that has been exposed to a SNCA inhibitor (e.g., a small molecule compound, a peptide, or a peptide mimetic) that inhibits SNCA activity, e.g., by inhibiting SNCA binding to a target protein (e.g., alpha-synuclein). Alternatively, the SNCA inhibitor can be a peptide or fragment derived from SNCA that acts in trans to inhibit SNCA activity. In some embodiments, the SNCA-modified DA neuron cell is irreversibly SNCA-inhibited, e.g., by genetic modification. In some embodiments, the SNCA-modified DA neuron cell is reversibly SNCA-inhibited, such that SNCA inhibition in the cell decreases over time.

[0261] As used herein, CIS inhibition refers to one or more of a decrease in net SNCA gene expression, net SNCA protein levels.

[0262] Sequence-specific reagents

[0263] A "sequence-specific reagent" refers to any active molecule that has the ability to specifically recognize a selected polynucleotide sequence at a genomic locus, given that the polynucleotide sequence is preferably at least 9 bp, more preferably at least 10 bp, even more preferably at least 12 pb in length, in view of modifying the genomic locus. In some embodiments, the sequence-specific reagent is preferably a sequence-specific nuclease reagent.

[0264] Target nucleic acid

[0265] As used herein, the term "target nucleic acid" or "target gene" refers to a nucleic acid that is being targeted for alteration, e.g., to generate a precise deletion, by the Cas system, Talen or ZNF system described herein. 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 can comprise one or more genes, e.g., 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.

[0266] Target position

[0267] As used herein, a "target position" refers to a site on a target nucleic acid (e.g., a chromosome) that is modified by a nuclease. For example, a target position can be modified by Cas protein molecule-mediated cleavage of a target nucleic acid and template nucleic acid-directed modification (e.g., correction) of the target position. In embodiments, a target position can be a site between two nucleotides (e.g., adjacent nucleotides) on a nucleic acid to which one or more nucleotides are added. A target position can comprise one or more nucleotides that are altered (e.g., corrected) by a template nucleic acid. In embodiments, a target position is within a "target sequence" (e.g., a sequence to which a gRNA binds). In embodiments, a target position is upstream or downstream of a target sequence (e.g., a sequence to which a gRNA binds).

[0268] Target position region

[0269] As used herein, a "target position region" is a region that comprises a target position. In certain embodiments, a target position is flanked by sequences of a target position region, i.e., the target position is disposed in a target position region such that there is target position region sequence both 5' and 3' of the target position. In certain embodiments, a target position region provides sufficient sequence on each side (i.e., 5' and 3') of a target position to allow gene conversion of the target position, where gene conversion uses an endogenous sequence homologous to the target position region as a template.

[0270] Target sequence

[0271] As used herein, a "target sequence" refers to a nucleic acid sequence that comprises a target position of a target gene.

[0272] Targeting domain

[0273] A "targeting domain" (which can alternatively be referred to as a guide sequence or a complementarity region) comprises, consists of, or consists essentially of a nucleic acid sequence that is complementary or partially complementary to a target nucleic acid sequence.

[0274] Template nucleic acid

[0275] A "template nucleic acid" when this term is used herein refers to a nucleic acid sequence that can be used in conjunction with a nuclease to alter the structure of a target location.

[0276] Therapeutic potential

[0277] "Therapeutic potential" reflects therapeutic activity as measured by in vitro experiments.

[0278] Sequence ID

[0279] SEQ ID No 1 : > Homo sapiens PARK2

[0280] SEQ ID No 2: > Homo sapiens PINK1

[0281] SEQ ID No 3: > Homo sapiens LRRK2

[0282] SEQ ID No 4: > Homo sapiens c-Rel

[0283] SEQ ID No 5: > Homo sapiens ATG7

[0284] SEQ ID No 6: > Homo sapiens VMAT2

[0285] SEQ ID No 7: > Homo sapiens GBA

[0286] SEQ ID No 8: > Homo sapiens SNCA

[0287] SEQ ID No 9: > Homo sapiens MAPT

[0288] SEQ ID No 10: > Homo sapiens GDNF

[0289] SEQ ID No 11 : > Homo sapiens PARK2

[0290] SEQ ID No 12: > Homo sapiens PINK1

[0291] SEQ ID No 13: > Homo sapiens LRRK2

[0292] SEQ ID No 14: > Homo sapiens c-Rel

[0293] SEQ ID No 15: > Homo sapiens ATG7

[0294] SEQ ID No 16: > Human VMAT2

[0295] SEQ ID No 17: > Human GBA

[0296] SEQ ID No 18: > Human SNCA

[0297] SEQ ID No 19: > Human MAPT

[0298] SEQ ID No 20: > Human GDNF

[0299] GDNF knock-in promoter

[0300] SEQ ID No 21: > Monoamine oxidase B (MAOB)

[0301] SEQ ID No 22: > GTP cyclohydrolase 1 (GCH1)

[0302] SEQ ID No 23: > Nuclear receptor subfamily 4, group A, member 2 (NR4A2)

[0303] SEQ ID No 24: > Mouse tyrosine hydroxylase (TH)

[0304] SEQ ID No 25: > Solute carrier family 6, member 3 (SLC6A3)

[0305] SEQ ID No 26: > Synapsin 1 (SYN1)

[0306] SEQ ID No 27: > Mouse alpha-Ca2+-calmodulin-dependent kinase II (Camk2a)

[0307] SEQ ID No 28: > Neurofilament medium chain (NEFM) mini-promoter

[0308] SEQ ID No 29: > Neurofilament light chain (NEFL) mini-promoter

[0309] SEQ ID No 30: > Neurofilament heavy chain (NEFH) promoter

[0310] GBA knock-in promoter

[0311] SEQ ID No 31: > Mouse Thy-1 cell surface antigen (mTHY1)

[0312] SEQ ID No 32: > Rat neuron-specific enolase (rNSE) promoter

[0313] SEQ ID No 33: > Dopacarboxylase (DDC) promoter

[0314] SEQ ID No 34: >Catechol-O-methyltransferase (COMT) promoter

[0315] SEQ ID No 35: >Glucosylceramidase beta 1 promoter 1 (GBA P1)

[0316] SEQ ID No 36: >Glucosylceramidase beta 1 promoter 2 (GBA P2)

[0317] SEQ ID No 37: >Human eukaryotic translation elongation factor 1 alpha 1 short form (EFS) promoter

[0318] SEQ ID No 38: >Human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter

[0319] SEQ ID No 39: >Chicken beta actin (CBA) promoter

[0320] SEQ ID No 40: >Cytomegalovirus (CMV) promoter

[0321] Protospacer adjacent motif (PAM)

[0322] SEQ ID No 41: NNGRRT

[0323] SEQ ID NO 42: NNGRRV

[0324] SNCA gene

[0325] The term“SNCA gene” encompasses the SNCA gene and functional fragments and variants thereof. A hemizygously null SNCA gene can be rendered hemizygously null via genetic engineering of the cell or via selection of the cell.

[0326] SNCA-modified DA neuronal cell

[0327] SNCA-modified DA neuronal cell refers to a DA neuronal cell that is SNCA+ / -.

[0328] Vector

[0329] A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. "Vectors" include, but are not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that can be comprised of chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acids. Preferred vectors are those that are capable of autonomous replication (episomal vectors) and / or expression of nucleic acids to which they are linked (expression vectors). A large number of suitable vectors are known to those of skill in the art, and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses (AAV)), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, noroviruses, togaviruses, flaviviruses, reoviruses, papillomaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis-sarcoma, mammalian type C, type B, type D viruses, HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0330] Compositions

[0331] 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 knock-in of the exogenous nucleic acid sequence knocks out the endogenous gene. 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 only expressed after the engineered cell is transplanted, i.e., not expressed during in vitro differentiation. In some embodiments, the endogenous gene comprises a promoter that is only expressed after the engineered cell is implanted, i.e., not expressed during in vivo differentiation.

[0332] DA neuronal cells with delayed GDNF expression

[0333] Disclosed herein are engineered DA neuronal cells that exhibit delayed expression of an exogenous GDNF gene in the engineered DA neuronal cells when administered to a patient, i.e., behind an endogenous promoter that does not express the exogenous GDNF gene after transplantation. In some embodiments, the exogenous GDNF gene is behind an endogenous promoter that is not active during in vitro differentiation of pluripotent cells into DA neuronal cells. In some embodiments, the exogenous GDNF gene is behind an endogenous promoter that is not active during in vivo differentiation of DA neuronal cells into neuronal mature cells. In some embodiments, the exogenous GDNF gene is behind a GDNF knock-in promoter selected from the group comprising or consisting of SEQ ID NOs: 21-30. In some embodiments, the exogenous GDNF gene is behind a GBA knock-in promoter selected from the group comprising or consisting of SEQ ID NOs: 31-40 or 43. In some embodiments, the delayed expression of the exogenous GDNF gene is delayed until implant function has been established. In some embodiments, the delayed expression of the exogenous GDNF gene is delayed until the transplanted engineered DA neuronal cells differentiate into neuronal mature cell types.

[0334] DA neuronal cells expressing GDB immediately and SNCA hemizygous null

[0335] Disclosed herein are engineered DA neuronal cells that, when administered to a patient, exhibit expression of an exogenous GBA gene and SCNA+ / - (exogenous or endogenous) gene in the engineered DA neuronal cells once the engineered DA neuronal cells are transplanted, i.e., the exogenous GBA gene is behind an endogenous promoter that is expressed post-transplant. In some embodiments, expression of the exogenous GBA gene occurs prior to the engineered DA neuronal cells having an implant function. In some embodiments, expression of the exogenous GBA gene occurs prior to the transplanted engineered DA neuronal cells differentiating into a mature neuronal cell type. In some embodiments, expression of the exogenous GBA gene occurs both prior to and after the transplanted engineered DA neuronal cells differentiating into a mature neuronal cell type. In some embodiments, a GDNF coding sequence is inserted under transcriptional control of an endogenous gene promoter that is not expressed until the administered engineered DA neuronal cells differentiate into a mature cell type in vivo. In some embodiments, the exogenous GBA gene is behind a GDNF knock-in promoter selected from the group comprising or consisting of SEQ ID NOs: 21-30. In some embodiments, the exogenous GBA gene is behind a GBA knock-in promoter selected from the group comprising or consisting of SEQ ID NOs: 31-40 or 43.

[0336] In some embodiments, the engineered DA neuronal cells are SNCA and / or MAPT hemizygous null that have been engineered to include an exogenous GBA gene. In some embodiments, the engineered DA neuronal cells are engineered to be SNCA and / or MAPT hemizygous null and in some embodiments, they are not engineered to be SNCA and / or MAPT hemizygous null.

[0337] In some embodiments, the engineered DA neuronal cells that have been engineered to include an exogenous GBA gene and are SNCA and / or MAPT hemizygous null have an altered ratio of GBA protein expression compared to alpha-synuclein and / or MAPT protein. In some embodiments, the ratio of GBA: alpha-synuclein protein expression in the administered engineered DA neuronal cells is higher. In some embodiments, the ratio of GBA: alpha-synuclein protein expression in the administered engineered DA neuronal cells is lower.

[0338] Engineered pluripotent cell populations

[0339] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous knock-in PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / - and / or GDNF gene or functional fragment thereof. In some embodiments, the exogenous knock-in gene is selected from the group comprising or consisting of SEQ ID NOs: 1-10.

[0340] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / - and / or GDNF gene or functional fragment thereof in a genomic safe harbor. In some embodiments, the exogenous gene is selected from the group comprising or consisting of SEQ ID NOs: 1-10. In some embodiments, the genomic safe harbor is selected from the group comprising or consisting of the safe harbors in Table 1.

[0341] Disclosed are engineered pluripotent cell populations, wherein the engineered DA neuronal cells comprise an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / - and / or GDNF gene or functional fragment thereof under the control of an endogenous gene promoter. In some embodiments, the endogenous gene promoter is selected from the group comprising or consisting of SEQ ID NOs: 21-30. In some embodiments, the endogenous gene promoter is selected from the group comprising or consisting of SEQ ID NOs: 31-40 or 43.

[0342] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous GDNF gene or functional fragment thereof. Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous SEQ ID NO: 10 or functional fragment thereof.

[0343] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SCNA and / or GDNF gene and an endogenous knock-out PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA and / or GDNF gene.

[0344] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous GDNF gene SEQ ID NO: 10 or a functional fragment thereof under the control of a promoter that is not expressed until an in vivo implant function has been established. Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous GDNF gene or a functional fragment thereof under the control of a promoter that is not expressed until the transplanted engineered pluripotent cell population differentiates in vivo into a mature neuronal cell type. Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous GDNF gene or a functional fragment thereof under the control of one of SEQ ID NOs: 21-40 or 43.

[0345] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cell populations comprise an exogenous knock-in GBA gene or a functional fragment thereof. Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous knock-in SEQ ID NO: 7 or a functional fragment thereof.

[0346] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous GBA gene or a functional fragment thereof under the control of a promoter that is expressed following cell transplantation in vivo. Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous GBA gene or a functional fragment thereof under the control of one of SEQ ID NOs: 21-40 or 43.

[0347] Disclosed are engineered pluripotent cells, wherein the engineered pluripotent cells comprise an exogenous GDNF gene or a functional fragment thereof under the control of a promoter that is not expressed until an in vivo implant function has been established, and further comprise an exogenous GBA gene or a functional fragment thereof under the control of a promoter that is expressed following cell transplantation.

[0348] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise (i) an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, and / or GDNF gene, and (ii) are haploinsufficient for a SNCA and / or MAPT gene.

[0349] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise (i) an exogenous GDNF gene or a functional fragment thereof, and (ii) are haploinsufficient for a SNCA and / or MAPT gene.

[0350] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise a disrupted SCNA and / or MAPT gene.

[0351] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise (i) an exogenous GBA gene or functional fragment thereof and (ii) a hemizygous null SNCA and / or MAPT gene.

[0352] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise (i) an exogenous GBA gene or functional fragment thereof and (ii) a hemizygous null SNCA gene.

[0353] In some embodiments, the engineered pluripotent cell populations do not express detectable levels of alpha-synuclein and / or MAPT protein in vivo and / or in vitro.

[0354] In some embodiments, the engineered pluripotent cell populations express less alpha-synuclein and / or MAPT protein in vivo and / or in vitro compared to wild-type pluripotent cells.

[0355] In some embodiments, the engineered pluripotent cell populations express less alpha-synuclein and / or MAPT protein in vivo and / or in vitro compared to engineered pluripotent cells without editing to the SNCA and / or MAPT gene.

[0356] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous SCNA gene.

[0357] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous SCNA+ / - gene.

[0358] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous SCNA gene, wherein the SCNA gene is located in a genomic safe harbor. In some embodiments, the SCNA gene is SCNA+ / +, SCNA+ / - or SCNA- / . In some embodiments, the genomic safe harbor is selected from the group comprising or consisting of the safe harbors in Table 1.

[0359] In some embodiments, the engineered pluripotent cell populations are human.

[0360] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous hemizygous null SNCA gene. Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous hemizygous null SNCA gene under the control of a promoter that is expressed after cell transplantation. Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous hemizygous null SNCA gene under the control of a promoter that is not expressed until after implant function has been established in vivo.

[0361] In some embodiments, the engineered pluripotent cell population comprising a hemizygous null SCNA gene comprises a disrupted SCNA gene. In some embodiments, the disrupted SCNA gene comprises the nucleotide sequence of SEQ ID NO: 8.

[0362] Engineered DA neuronal cell populations

[0363] Disclosed are engineered DA neuronal cell populations, wherein the engineered DA neuronal cells comprise an exogenous knock-in PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / - and / or GDNF gene or functional fragment thereof. Disclosed are engineered DA neuronal cell populations, wherein the engineered DA neuronal cells comprise an exogenous knock-in gene selected from the group comprising or consisting of SEQ ID NOs: 1-10 or functional fragments thereof.

[0364] Disclosed are engineered DA neuronal cell populations, wherein the engineered DA neuronal cells comprise an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / - and / or GDNF gene or functional fragment thereof in a genomic safe harbor. In some embodiments, the genomic safe harbor is selected from the group comprising or consisting of the safe harbors in Table 1.

[0365] Disclosed are engineered DA neuronal cell populations, wherein the engineered DA neuronal cells comprise an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / - and / or GDNF gene or functional fragment 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.

[0366] Disclosed are engineered DA neuronal cell populations, wherein the engineered DA neuronal cells comprise an exogenous GDNF gene or functional fragment thereof.

[0367] Disclosed are engineered pluripotent cell populations, wherein the engineered pluripotent cells comprise an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SCNA and / or GDNF gene and an endogenous knock-out PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA and / or GDNF gene.

[0368] Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise an exogenous GDNF gene or functional fragment thereof under the control of a promoter that is not expressed until a portion of the transplanted DA neuron cell population has engrafted in vivo. Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise an exogenous GDNF gene or functional fragment thereof under the control of a promoter that is not expressed until the transplanted engineered DA neuron cell population has differentiated in vivo to a neuronal mature cell type.

[0369] Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise an exogenous GBA gene or functional fragment thereof. Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise an exogenous SEQ ID NO: 7 or functional fragment thereof.

[0370] Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise an exogenous GBA gene or functional fragment thereof under the control of a promoter that is expressed after cell transplantation. In some embodiments, the endogenous gene promoter is one of SEQ ID NOs: 21-40 or 43.

[0371] Disclosed are engineered DA neuron cells, wherein the engineered DA neuron cells comprise an exogenous GDNF gene or functional fragment thereof under the control of a promoter that is not expressed until engraftment function has been established in vivo, and further comprise an exogenous GBA gene or functional fragment thereof under the control of a promoter that is expressed after cell transplantation.

[0372] Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise (i) an exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, and / or GDNF gene, and (ii) are hemizygous null for a SNCA and / or MAPT gene.

[0373] Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise (i) an exogenous GDNF gene or functional fragment thereof and (ii) are hemizygous null for a SNCA and / or MAPT gene.

[0374] Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise a disrupted SCNA and / or MAPT gene.

[0375] Disclosed are engineered DA neuron cell populations, wherein the engineered DA neuron cells comprise (i) an exogenous GBA gene or functional fragment thereof and (ii) are hemizygous null for a SNCA and / or MAPT gene.

[0376] Disclosed is an engineered DA neuron cell population, wherein the engineered DA neuron cell comprises (i) an exogenous GBA gene or functional fragment thereof and (ii) a hemizygous null SNCA gene.

[0377] In some embodiments, the engineered DA neuron cell population does not express detectable levels of alpha-synuclein and / or MAPT protein in vivo and / or in vitro.

[0378] In some embodiments, the engineered DA neuron cell population expresses less alpha-synuclein and / or MAPT protein compared to a wild-type DA neuron cell.

[0379] In some embodiments, the engineered DA neuron cell population expresses less alpha-synuclein and / or MAPT protein in vivo and / or in vitro compared to an engineered DA neuron cell without editing to the SNCA and / or MAPT gene.

[0380] Disclosed is an engineered pluripotent cell population, wherein the engineered pluripotent cell comprises an exogenous SCNA gene, wherein the SCNA gene is located in a genomic safe harbor. In some embodiments, the SCNA gene is SCNA+ / +, SCNA+ / - or SCNA- / . In some embodiments, the genomic safe harbor is selected from the group comprising or consisting of the safe harbors in Table 1.

[0381] In some embodiments, the engineered DA neuron cell population is human. In some embodiments, the engineered DA neuron cell population is derived from a pluripotent cell.

[0382] Disclosed is an engineered DA neuron cell population, wherein the engineered DA neuron cell comprises an exogenous hemizygous null SNCA gene. Disclosed is an engineered DA neuron cell population, wherein the engineered DA neuron cell comprises an exogenous hemizygous null SNCA gene under the control of a promoter that is expressed after cell transplantation. Disclosed is an engineered DA neuron cell population, wherein the engineered DA neuron cell comprises an exogenous hemizygous null SNCA gene under the control of a promoter that is not expressed until function of an in vivo implant has been established.

[0383] In some embodiments, the engineered DA neuron cell population comprising a hemizygous null SNCA gene comprises a disrupted SCNA gene. In some embodiments, the disrupted SCNA gene comprises the nucleotide sequence of SEQ ID NO: 8.

[0384] In some embodiments, at least 5-95% of the engineered DA neuronal cells do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 20-75% of the engineered DA neuronal cells do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 30% of the engineered DA neuronal cells do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 50% of the engineered DA neuronal cells do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 70% of the engineered DA neuronal cells do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 80% of the engineered DA neuronal cells do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 90% of the engineered DA neuronal cells do not express detectable levels of alpha-synuclein protein.

[0385] In some embodiments, the GBA gene is under the control of a promoter that is expressed upon transplantation of the engineered cells into the patient. In some embodiments, the SCNA gene is under the control of a promoter that is expressed upon transplantation of the engineered cells into the patient, but to a lesser extent than the GBA gene promoter.

[0386] Disclosed are DA neuronal cells that promote expression of GDNF by site-directed gene editing. Disclosed are DA neuronal cells that promote expression of GBA by site-directed gene editing. Disclosed are DA neuronal cells that promote expression of GBA by site-directed gene editing and modulate expression of SNCA. Disclosed are DA neuronal cells that modulate expression of SNCA by site-directed gene editing. Disclosed are DA neuronal cells that promote expression of GBA by site-directed gene editing and are SNCA hemizygous null. Disclosed are DA neuronal cells that promote expression of GBA by site-directed gene editing and are SNCA hemizygous null.

[0387] Disclosed are DA neuronal cells that promote expression of GDNF and GBA by site-directed gene editing and are SNCA hemizygous null. In some embodiments, GDNF expression is delayed until implant formation has been established. In some embodiments, the GBA gene is expressed upon transplantation of the engineered DA neuronal cells. In some embodiments, the GBA gene is expressed and SNCA gene expression is modulated upon transplantation of the engineered DA neuronal cells, wherein SNCA protein expression is less than GBA protein expression. In some embodiments, GDNF expression is promoted after implant formation has been established, and the GBA gene is expressed upon transplantation of the engineered DA neuronal cells.

[0388] 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 SEQ ID NOs: 21-40 or 43.

[0389] In some embodiments, the exogenous sequence is integrated at an endogenous locus that constitutively expresses / constitutively transcribes.

[0390] In some embodiments, it can be advantageous to inactivate the endogenous SNCA coding sequence while transcribing an integrated exogenous sequence encoding a GBA, SNCA+ / -, or GDNF gene or functional fragment thereof at the locus.

[0391] In some embodiments, SNCA gene expression in the transplanted engineered DA neuron cell is prevented or reduced compared to wild type.

[0392] The embodiments disclosed herein can be further understood by the following numbered paragraphs:

[0393] Paragraph 1. A population of DA neuron cells having the following features:

[0394] an exogenous sequence that has been integrated under the transcriptional control of an endogenous gene promoter.

[0395] Paragraph 2. The engineered DA neuron cell according to Paragraph 1 having the genotype [SNCA] neg [GBA] pos .

[0396] Paragraph 3. The engineered DA neuron cell according to Paragraph 1 having the genotype [SNCA] neg [GBA] pos [GDNF] pos .

[0397] Paragraph 4. The engineered DA neuron cell according to Paragraph 1 having the genotype [SNCA] + / - [GBA] pos [GDNF] pos .

[0398] Paragraph 5. A population of DA neuron cells having the following features:

[0399] an exogenous GDNF, GBA, and / or SNCA sequence that has been integrated under the transcriptional control of an endogenous gene promoter.

[0400] Paragraph 6. A therapeutically effective population of DA neuronal cells comprising at least 30%, preferably at least 50%, more preferably at least 80% of engineered DA neuronal cells according to any one of paragraphs 1 to 5.

[0401] Type of exogenous sequence

[0402] In some embodiments, the exogenous sequence added to the cell (pluripotent or DA neuronal cell) to be engineered is a polynucleic acid. In some embodiments, the polynucleic acid is DNA or RNA. In some embodiments, the RNA can be mRNA. Also disclosed herein are exogenous polynucleic acid sequences comprising at least one exogenous GDNF, GBA, or SNCA sequence. The exogenous polynucleic acid sequence can be complementary to a genomic sequence, which can be a partial sequence or a complete sequence. In some embodiments, the exogenous polynucleic acid sequence comprises a coding sequence. In some embodiments, the exogenous polynucleic acid sequence comprises a non-coding sequence. In some embodiments, the exogenous polynucleic acid sequence comprises one or more genes.

[0403] In some embodiments, the polynucleic acid can be a plasmid vector. The plasmid vector can comprise a promoter. In some embodiments, the promoter can be constitutive. In some embodiments, the promoter can be inducible. In some embodiments, the promoter is synapsin 1, DAT, VMAT, TH, AADC, tamoxifen inducible promoter, RU486 inducible promoter, or other promoters that allow for temporal control or small molecule control. In some embodiments, the promoter can be adjacent to the exogenous GDNF, GBA, and / or SNCA sequence. In some embodiments, the plasmid vector further comprises a splice acceptor. In some embodiments, the splice acceptor can be adjacent to the exogenous GDNF, GBA, and / or SNCA sequence.

[0404] In some embodiments, the plasmid vector further comprises a translation initiation codon, such as an “ATG” sequence. The translation initiation codon sequence can be adjacent to the GDNF, GBA, and / or SNCA sequence. In some embodiments, the GDNF, GBA, and / or SNCA sequence can be within a polycistronic vector. In some embodiments, the polynucleic acid comprises an exogenous promoter, an endogenous promoter via splicing, and / or an endogenous promoter via in-frame translation.

[0405] In some embodiments, the plasmid can be modified. The modification can comprise demethylation, addition of CpG methylation, removal of bacterial methylation, and addition of mammalian methylation.

[0406] Embodiments can be further understood by the following numbered paragraphs:

[0407] Paragraph 1. An engineered DA neuronal cell comprising an exogenous PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene or functional fragment thereof.

[0408] Paragraph 2. A composition comprising the engineered DA neuronal cell of paragraph 1.

[0409] Paragraph 3. A genome editing system comprising:

[0410] (a) a gRNA molecule;

[0411] (b) a Cas molecule configured to alter a PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF gene.

[0412] Paragraph 4. A method of inhibiting degeneration or death of a dopaminergic neuron, comprising:

[0413] contacting a neuron with the engineered DA neuronal cell of paragraph 1;

[0414] wherein upon contact with the engineered DA neuronal cell, the neuron expresses the PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, SNCA, and / or GBA protein,

[0415] optionally, wherein the neuron comprises a mutation in the PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, SNCA, and / or GBA gene.

[0416] Paragraph 5. The method of paragraph 4, wherein the neuron expresses a reduced amount of alpha-synuclein upon contact with the engineered DA neuronal cell.

[0417] Paragraph 6. The method of any one of paragraphs 4-5, wherein the neuron produces and / or releases an increased amount of dopamine upon contact with the engineered DA neuronal cell.

[0418] Paragraph 7. The method of any one of paragraphs 4-6, wherein the neuron expresses a lower amount of alpha-synuclein compared to the amount of alpha-synuclein expressed in a neuron that has not been contacted with an engineered DA neuronal cell, optionally wherein 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 the neuron that has not been contacted with the engineered DA neuronal cell.

[0419] Paragraph 8. The method of any one of paragraphs 4 to 7, wherein the neurons produce and / or release an increased amount of dopamine compared to the amount of dopamine produced and / or released by neurons that are not in contact with the engineered DA neuronal cells, optionally wherein 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 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater than the amount produced and / or released by neurons that are not in contact with the engineered DA neuronal cells.

[0420] Paragraph 9. The method of any one of paragraphs 4 to 8, wherein the neurons undergo an increased amount of autophagy compared to the amount of autophagy experienced by neurons that are not in contact with the engineered DA neuronal cells, optionally wherein 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 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater than the amount experienced by neurons that are not in contact with the engineered DA neuronal cells.

[0421] Paragraph 10. The method of any one of paragraphs 4 to 9, wherein the neurons are primary tyrosine hydroxylase positive neurons.

[0422] Paragraph 11. A method of treating or inhibiting the onset of Parkinson’s disease in a subject having or at risk of Parkinson’s disease (PD), comprising:

[0423] administering to the subject the engineered DA neuronal cells of paragraph 1;

[0424] wherein administration of the engineered DA neuronal cells treats or inhibits the onset of Parkinson’s disease in the subject.

[0425] Paragraph 12. The method of claim 11, wherein the subject is an adult or a child.

[0426] Paragraph 13. The method of any one of paragraphs 11 to 12, wherein the number of dopaminergic neurons in the subject after the administering step is greater than the number of dopaminergic neurons in the subject before the administering step.

[0427] Paragraph 14. The method of any one of paragraphs 11 to 13, wherein the level of dopamine in the subject after the administering step is greater than the level of dopamine in the subject before the administering step.

[0428] Paragraph 15. The method of any one of paragraphs 11 to 14, wherein the number of dopaminergic neurons in the subject treated by the method is increased compared to the number of dopaminergic neurons in a subject that is not so treated.

[0429] Paragraph 18. The method of any one of paragraphs 11 to 17, wherein the level of GDNF and / or GBA in the CSF of the subject after the administering step is greater than the level of GDNF and / or GBA in the CSF of the subject before the administering step.

[0430] Paragraph 17. The method of any one of paragraphs 11 to 16, wherein the level of dopamine in the substantia nigra of the subject treated by the method is increased compared to the level of dopamine in the substantia nigra of a subject not so treated.

[0431] Paragraph 18. The method of any one of paragraphs 11 to 17, wherein the level of GDNF and / or GBA in the CSF of the subject after the administering step is greater than the level of GDNF and / or GBA in the CSF of the subject before the administering step.

[0432] Paragraph 19. The method of any one of paragraphs 11 to 18, wherein the Unified Parkinson’s Disease Rating Scale (UPDRS) score of the subject before the administering step is improved compared to the UPDRS score of the subject before the administering step.

[0433] Paragraph 20. The method of any one of paragraphs 11 to 19, wherein the level of PRKN in the CSF of the subject treated by the method is increased compared to the level of PRKN in the CSF of a subject not so treated.

[0434] Paragraph 21. The method of any one of paragraphs 11 to 19, wherein the Unified Parkinson’s Disease Rating Scale (UPDRS) score of the subject treated by the method is improved compared to the UPDRS score of a subject not so treated.

[0435] Paragraph 22. The method of any one of paragraphs 11 to 21, wherein the neurons of the subject express a reduced amount of alpha-synuclein and / or comprise a reduced amount of Lewy bodies after contact with the engineered DA neuronal cells.

[0436] Paragraph 23. A population of DA neuronal cells comprising a recombinant genetic vector, the recombinant genetic vector comprising a polynucleotide encoding a wild-type PINK1, LRRK2, SNCA, c-Rel, ATG7, VMAT2, GDNF, or GBA gene, a ubiquitin-like modifier activating enzyme (ATG7) gene, a synaptic vesicle amine transport protein (VMAT2) gene, or a glucocerebrosidase (GBA) gene, a GDNF gene, a SNCA + / - gene, or a functional variant or fragment thereof; wherein the polynucleotide is operably linked to a eukaryotic active promoter.

[0437] Cell types to be engineered

[0438] In some embodiments, the cell to be engineered can be a primary cell. The primary cell can be a neural cell, a pluripotent cell, a progenitor cell, or a combination thereof. In some embodiments, the progenitor cell is a DA neuronal cell (see Figure 1 Markers for different neural lineage cell types). Examples of pluripotent cells include stem cells, such as umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (IPSs). The engineered cell can be a human cell. The engineered cell can be an animal (non-human cell). The engineered cell can be expanded ex vivo. The engineered cell can be expanded in vitro. The engineered cell can be expanded in vivo. The engineered cell can be autologous to a subject in need thereof. The engineered cell can be non-autologous to a subject in need thereof. The engineered cell can be in vitro. The engineered cell can be in vivo. The engineered cell can be part of a combination therapy to treat Parkinson’s disease and other and secondary parkinsonian disorders in a subject in need thereof.

[0439] Endogenous gene disrupted by exogenous gene sequence

[0440] In some embodiments, the endogenous gene is disrupted by an 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 a wild-type GDNF, GBA, and / or SNCA gene. In some embodiments, the endogenous gene is a mutated GDNF, GBA, and / or SNCA gene. In some embodiments, the endogenous gene is a mutated PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / - and / or GDNF gene. In some embodiments, the endogenous gene can be under the control of a promoter. In some embodiments, the exogenous gene can be under the control of a promoter selected from the group comprising synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, RU486-inducible promoter, and / or other promoters that allow for temporal control or small molecule control. In some embodiments, the endogenous gene can be under the control of a promoter. In some embodiments, the exogenous gene can be under the control of a promoter selected from the group comprising synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, or RU486-inducible promoter.

[0441] Engineered with more than one exogenous gene sequence

[0442] In some embodiments, the engineered cell can comprise a single GDNF, GBA, and / or SNCA exogenous sequence. In some embodiments, the engineered cell can comprise multiple GDNF, GBA, and / or SNCA exogenous sequences. In some embodiments, the engineered cell can comprise more than one of a GDNF, GBA, or SNCA exogenous sequence. The GDNF exogenous sequence can comprise an engineered GDNF exogenous sequence. The GDNF exogenous sequence can produce a functional GDNF protein. The GBA exogenous sequence can comprise an engineered GBA exogenous sequence. The GBA exogenous sequence can produce a functional GBA protein. The SCNA exogenous sequence can comprise an engineered SCNA exogenous sequence. The SNCA exogenous sequence can produce a functional SCNA protein.

[0443] protospacer adjacent motif sequence

[0444] Disclosed herein can be engineered cells comprising at least one exogenous GDNF, GBA, and / or SNCA gene or functional fragment thereof, which can be adjacent to a protospacer adjacent motif sequence of genomic DNA. In some embodiments, the protospacer adjacent motif (PAM) can be recognized by a CRISPR endonuclease. The endonuclease can be a Cas protein. The Cas protein can be selected from the list comprising: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csy l, Csy2, Csy3, Cse l, Cse2, Csc l, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr l, Cmr3, Cmr4, Cmr5, Cmr6, Csb l, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csx l, Csx IS, Csf l, Csf2, CsO, Csf4, Cpf l, c2c l, c2c3, Cas9HiFi, homologs thereof, or modified versions thereof. In some embodiments, the CRISPR endonuclease can be Cas9. The Cas9 disclosed herein can recognize a PAM sequence, which can be 5’NGG 3’.

[0445] Disclosed herein can be at least one exogenous GDNF that can disrupt at least one gene. The disrupted gene can be any of the genes as set forth in SEQ ID NOs: 1-10. Disclosed herein can be at least one exogenous GBA that can disrupt at least one gene. The disrupted gene can be any of the genes as set forth in SEQ ID NOs: 1-10. Disclosed herein can be at least one exogenous SNCA that can disrupt at least one gene. The disrupted gene can be any of the genes as set forth in SEQ ID NOs: 1-10. In some embodiments, the disrupted gene can comprise a protospacer sequence. The protospacer sequence can be disrupted by insertion of an exogenous gene sequence. The GDNF gene sequence can produce functional GDNF. The GBA gene sequence can produce functional GBA. The SNCA gene sequence can produce functional SNCA.

[0446] Disclosed herein can be a composition comprising at least one guide RNA that binds to an endogenous GDNF, GBA, or SNCA gene and at least one secondary guide RNA that binds to an endogenous gene selected from the group comprising or consisting of any of the genes as set forth in SEQ ID NOs: 1-10.

[0447] Disclosed herein can be an engineered cell having a disruption in an 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 that is not PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF.

[0448] Disclosed herein are a genetically modified cell derived from a human subject; a multi-nucleic acid that targets a multi-nucleic acid, wherein the multi-nucleic acid that targets a multi-nucleic acid is engineered to hybridize to a specific region of a target gene in the genome of the cell; a nuclease, wherein the nuclease is capable of binding to the multi-nucleic acid that targets a multi-nucleic acid to form a nucleoprotein complex, wherein the nucleoprotein complex can be capable of generating a targeted double-strand break in the target gene in the genome of the cell; and a target multi-nucleic acid, wherein the target multi-nucleic acid can be genomic DNA comprising a double-strand break in the target gene, wherein the double-strand break in the target gene results in a disruption of the function of the target gene, and wherein the genetically modified cell can be capable of being expanded to generate a clonal population of cells having an altered function of the target gene, and wherein the clonal population of modified cells is suitable for administration to a human in need thereof.

[0449] Gene editing system

[0450] The genome editing systems disclosed herein include at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex that is capable of binding to a specific nucleic acid sequence in a cell and editing the DNA in or around that nucleic acid sequence, for example by creating one or more of a single-stranded break (SSB or nick), a double-stranded break (DSB), and / or a point mutation. In various embodiments, a genome editing system can comprise (a) one or more Cas9 / gRNA complexes, and (b) separate Cas9 molecules and gRNAs that are capable of binding in a cell to form one or more Cas9 / gRNA complexes. Genome editing systems according to the present disclosure can be encoded by one or more nucleotides (e.g., RNA, DNA) comprising coding sequences for Cas9 and / or gRNAs that can bind to form a Cas9 / gRNA complex, and one or more nucleotides encoding a gene editing system can be carried by a vector as described herein.

[0451] In certain embodiments, the genome editing system targets a neuronal gene selected from the group comprising or consisting of PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF. In certain embodiments, the genome editing system targets a neuronal gene selected from the group comprising or consisting of SEQ ID NOs: 1-10.

[0452] The present disclosure relates to a guide RNA (gRNA) comprising a targeting domain comprising 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 configured to bind to a target nucleotide sequence set forth 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 a complement or RNA equivalent thereof. In certain embodiments, the targeting domain is 3 to 100, 5 to 100, 10 to 100, or 20 to 100 nucleotides in length, and in certain of these embodiments, the targeting domain is 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 length. In certain embodiments, the targeting domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. Methods for selecting a targeting domain are known in the art (see, e.g., Fu et al. (2014) NAT. BIOTECHNOL. 32(3): 279-84; Sternberg et al. (2014) NATURE 507(7490): 62-67, the entire contents of each of which are expressly incorporated herein by reference). Since the targeting domain is part of a gRNA molecule, it comprises a base uracil (U) rather than thymine (T); conversely, any DNA molecule encoding a gRNA molecule can comprise thymine rather than uracil. In a targeting domain / target domain pair, the uracil base in the targeting domain can pair with an adenine base in the target domain. In certain embodiments, the degree of complementarity between the targeting domain and the target domain is sufficient to allow a Cas9 molecule to target a target nucleic acid. Thus, a targeting domain or gRNA described herein includes a portion of SEQ ID NO: 1-10 that is 3 to 100 nucleotides in length and comprises a base uracil (U) rather than thymine (T). In certain embodiments, the targeting domain is fully complementary to a target domain. In certain embodiments, the targeting domain is fully complementary to SEQ ID NO: 1-10 and comprises a base uracil (U) rather than thymine (T).Likewise, where the targeting domain comprises 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 portion of SEQ ID NOs: 1-10.

[0453] 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 to the corresponding portion of the target domain. In certain embodiments, the targeting domain includes one or more nucleotides that are not complementary to the target domain, and in certain embodiments of these embodiments, the targeting domain includes 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that are not complementary to the target domain. In certain embodiments where the targeting domain includes one or more nucleotides that are not complementary to the target domain, one or more of the non-complementary nucleotides is within five nucleotides of the 5’ or 3’ end of the targeting domain. In certain embodiments of these embodiments, the targeting domain includes 1, 2, 3, 4, or 5 nucleotides that are not complementary to the target domain within 5 nucleotides of the 5’ end, the 3’ end, or both the 5’ end and the 3’ end thereof. In certain embodiments where the targeting domain includes two or more nucleotides that are not complementary to the target domain, two or more of the non-complementary nucleotides are adjacent to one another, and in certain embodiments of these embodiments, two or more contiguous non-complementary nucleotides are within five nucleotides of the 5’ or 3’ end of the targeting domain. In other embodiments, both of the two or more contiguous non-complementary nucleotides are more than five nucleotides from the 5’ and 3’ ends of the targeting domain.

[0454] In certain embodiments, the targeting domain consists of, consists essentially of, or comprises 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 contiguous nucleotides) that are complementary or partially complementary to the target domain or a portion thereof, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certain embodiments of these embodiments, the targeting domain is complementary to the target domain over the entire length of the targeting domain, the entire length of the target domain, or both.

[0455] In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the first 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10 (residues 1-20). In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the second 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10 (residues 20-40). In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the third 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10 (residues 40-60). In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the fourth 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10 (residues 60-80). In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the fifth 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10 (residues 80-100). In some embodiments, the targeting domain is listed as 20 N (residues 1-20), but can range in length from 16 to 26 nucleotides.

[0456] In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the last 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10. In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the next to last 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10. In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the third to last 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10. In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the fourth to last 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10. In certain embodiments, the single molecule or chimeric gRNA molecules disclosed herein (comprising a targeting domain) comprise the fifth to last 20 N of the amino acid sequence set forth in SEQ ID NO: 1-10.

[0457] In certain embodiments, the modification of one or more nucleotides in the targeting domain or the non-complementary nucleotides does not interfere with targeting efficacy, which can be assessed by testing 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 assessed using systems known in the art. The candidate targeting domain can be placed alone or with one or more other candidate changes known to be functional for the selected target in a gRNA molecule / Cas9 molecule system and assessed.

[0458] The present disclosure also relates to a DNA targeting composition comprising a first gRNA and a second gRNA. The first gRNA molecule and the second gRNA molecule comprise a targeting domain comprising 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 configured to bind to a target nucleotide sequence set forth 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 a complement thereof. In some embodiments, the first gRNA molecule and the second gRNA molecule comprise different targeting domains.

[0459] The present disclosure also relates to an isolated polynucleotide comprising a gRNA molecule described above or a DNA targeting composition described above.

[0460] The present disclosure relates to a vector comprising a gRNA described above, a DNA targeting composition described above, or an isolated polynucleotide described above.

[0461] The present disclosure also relates to a vector comprising a DNA targeting composition described above.

[0462] The present disclosure also relates to a vector encoding: (a) a first guide RNA (gRNA) molecule, (b) a second gRNA molecule, and (c) at least one Cas9 molecule recognizing a protospacer adjacent motif (PAM) of NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42). The first gRNA molecule and the second gRNA molecule comprise a targeting domain that 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, or SEQ ID NO: 10 (or a portion thereof), or is configured to bind to a target nucleotide sequence set forth 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 a complement thereof. In some embodiments, the first gRNA molecule and the second gRNA molecule comprise different targeting domains.

[0463] The present disclosure also relates to a cell comprising the above-described gRNA, the above-described DNA-targeting composition, the above-described isolated polynucleotide, and / or the above-described vector.

[0464] The present disclosure also relates to a kit comprising the above-described gRNA, the above-described DNA-targeting system, the above-described isolated polynucleotide, the above-described vector, or the above-described cell, and optionally instructions for use.

[0465] The present disclosure also relates to a method of correcting a mutant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA gene in a cell. The method comprises administering to the cell the above-described gRNA, the above-described DNA-targeting system, the above-described isolated polynucleotide, or the above-described vector.

[0466] The present disclosure also relates to a method of genome editing a mutant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA gene in a subject. The method comprises administering to the subject a genome editing composition comprising the above-described gRNA, the above-described DNA-targeting system, the above-described isolated polynucleotide, the above-described vector, or the above-described cell.

[0467] The present disclosure also relates to a method of treating a subject in need thereof having a mutant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA gene. The method comprises administering to the subject the gRNA described above, the DNA-targeting system described above, the isolated polynucleotide described above, the vector described above, or the cell described above.

[0468] The present disclosure also relates to a modified adeno-associated viral vector for genome editing of a mutant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA gene in a subject, comprising a first polynucleotide sequence encoding the gRNA described above and a second polynucleotide sequence encoding a Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42).

[0469] The present disclosure also relates to a cell comprising the composition described above.

[0470] A vector encoding a guide RNA (gRNA) molecule and a Cas9 molecule, wherein the gRNA molecule comprises a targeting domain comprising 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 is configured to bind to a target nucleotide sequence set forth 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 a complement thereof.

[0471] A genome editing system comprising: (a) a gRNA molecule comprising a targeting domain configured to bind to a target nucleotide sequence set forth 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 a complement thereof; and (b) a Cas9 molecule.

[0472] Non-limiting methods for assessing chromatin accessibility include micrococcal nuclease (MNase)-assisted nucleosome isolation sequencing (MNase-seq), DNase I hypersensitive site sequencing (DNase-seq), formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq), and transposase accessible chromatin sequencing assay (ATAC-seq). In some embodiments, chromatin accessibility is determined by high-throughput transposase accessible chromatin sequencing assay (ATAC-seq). In some embodiments, the disclosed methods further comprise selecting a locus as a GSH if the locus is located at a distance of at most about 10 kb, at most about 9 kb, at most about 8 kb, at most about 7 kb, at most about 6 kb, at most about 5 kb, at most about 4 kb, at most about 3 kb, at most about 2 kb, or at most about 1 kb from an ATAC-seq peak or within an ATAC-seq peak.

[0473] The gene editing systems disclosed herein can be further understood by the numbered paragraphs below:

[0474] Paragraph 1. A gene editing system for a cell, comprising:

[0475] a. a Cas protein or a polynucleotide encoding a Cas protein;

[0476] b. a guide RNA (gRNA); and

[0477] c. a repair template comprising a functional PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA gene or a functional variant or fragment thereof;

[0478] wherein the gene editing system is capable of repairing an endogenous gene in the cell or inserting a functional gene into the genome of the cell in vivo or in vitro.

[0479] Paragraph 2. The gene editing system of Paragraph 1, wherein at least one component of the gene editing system is delivered by a recombinant AAV.

[0480] Paragraph 3. The gene editing system of Paragraph 1, wherein the gene editing system is delivered by a recombinant AAV.

[0481] Paragraph 4. The gene editing system of any one of Paragraphs 1-3, wherein the cell is an in vitro pluripotent cell or a DA neuronal cell.

[0482] Paragraph 5. The gene editing system of any one of Paragraphs 1-3, wherein the cell is an in vivo dopaminergic neuron.

[0483] Recombinant gene vectors

[0484] Various embodiments and methods related to recombinant genetic vectors comprising PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, and / or GBA or a functional fragment or variant thereof are disclosed. In some embodiments, the vector further comprises an edit to the alpha-synuclein (SCNA) gene to render the population of cells into which the vector is introduced SNCA hemizygous null. In some embodiments, the recombinant genetic vector renders the cell into which it is introduced SNCA homozygous null and / or MAPT homozygous null. In some embodiments, the recombinant genetic vector renders the cell into which it is introduced SNCA hemizygous null and / or MAPT homozygous null. In some embodiments, the recombinant genetic vector renders the cell into which it is introduced SNCA homozygous null and / or MAPT hemizygous null. In some embodiments, the recombinant genetic vector does not include an SCNA gene edit.

[0485] Various embodiments and methods related to recombinant genetic vectors comprising a GDNF gene (SEQ ID NO: 10) or a functional fragment or variant thereof are disclosed. In some embodiments, the vector further comprises an edit to the GBA gene. In some embodiments, the vector further comprises an edit to the SNCA gene.

[0486] Various embodiments and methods related to recombinant genetic vectors comprising a GBA gene (SEQ ID NO: 7) or a functional fragment or variant thereof are disclosed. In some embodiments, the vector further comprises an edit to the SNCA gene.

[0487] Various viral or non-viral vectors can be used. In some embodiments, the recombinant genetic vector is a recombinant adeno-associated virus (AAV). Any known serotype can be used. In some embodiments, the AAV has a serotype of AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. In some embodiments, the recombinant genetic vector comprises a self-complementary AAV. In some embodiments, the recombinant genetic vector comprises a single-stranded AAV. In some embodiments, the AAV is a wild-type AAV or a modified AAV. In some embodiments, the AAV comprises a capsid protein that is at least 95% identical to a wild-type VP1, VP2, or VP3 capsid protein.

[0488] The recombinant genetic vector can include a gene regulatory element. In some embodiments, the recombinant genetic vector comprises a polynucleotide comprising, in the following 5’ to 3’ order, a eukaryotic active promoter sequence and a sequence encoding a wild-type protein, or a functional fragment or variant thereof. The sequence encoding a wild-type protein, or a functional fragment or variant thereof, is operably linked to the eukaryotic active promoter sequence.

[0489] In some embodiments, the vectors and methods disclosed herein, the vector comprises an expression cassette comprising, in 5' to 3' order:

[0490] a promoter (GDNF knock-in promoter [such as MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoter [such as 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 for temporal control or small molecule control), a gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF);

[0491] a promoter (GDNF knock-in promoter [such as MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoter [such as 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), a gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF);

[0492] a promoter (GDNF knock-in promoter [such as MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoter [such as 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 for temporal control or small molecule control), a gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF), and is hemizygously null for the SNCA and / or MAPT gene;

[0493] 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 for temporal or small molecule control), 5' enhancer, gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF).

[0494] 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 for temporal or small molecule control), 5' enhancer, gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF), and is hemizygously null for the SNCA and / or MAPT gene.

[0495] In some embodiments, the recombinant genetic vector comprises one or more of a neuron-specific promoter, optionally selected from the group comprising or consisting of: hSYN1 (human synapsin), INA (alpha-filamin), NES (nestin), TH (tyrosine hydroxylase), FOXA2 (Forkhead box A2), CaMKII (calmodulin-dependent protein kinase II), and NSE (neuron-specific enolase) promoters.

[0496] In some embodiments, the recombinant genetic vector comprises a promoter selected from the group comprising or consisting of: CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, and chicken beta-actin promoters.

[0497] In some embodiments, the engineered DA neuronal cells disclosed transduced with a recombinant genetic vector further comprise a mutation in a gene associated with Parkinson's Disease (PD). The mutated gene can be SNCA and / or MAPT. The mutated gene can be one of SEQ ID NOs: 1-10 or a combination thereof.

[0498] Disclosed are vectors encoding a guide RNA (gRNA) molecule and a Cas protein molecule, wherein the gRNA molecule comprises a targeting domain comprising a portion of a nucleotide sequence selected from SEQ ID NOs: 1-10. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector. In some embodiments, the vector is a nucleic acid sequence containing an origin of replication. In some embodiments, the vector can 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, and preferably a DNA plasmid. For example, the vector can encode a Cas9 protein and at least one gRNA molecule. In some embodiments, the Cas9 protein can be a Staphylococcus aureus Cas9, such as SaCas9.

[0499] Disclosed are genome editing systems comprising: (a) a gRNA molecule comprising a targeting domain comprising a portion of a nucleotide sequence selected from SEQ ID NOs: 1-10, (b) a Cas protein molecule.

[0500] In some embodiments, the vector or genome editing system is configured to alter a GDNF, GBA, or SNCA gene. In some embodiments, the vector or genome editing system is configured to alter SEQ ID NO: 10, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the Cas protein molecule is a Staphylococcus aureus Cas9 molecule. In some embodiments, the Cas protein molecule recognizes a protospacer adjacent motif (PAM) of NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42).

[0501] The presently disclosed subject matter also provides cells comprising the vectors or genome editing systems described herein. In some embodiments, the cells are pluripotent cells or DA neuronal cells or dopaminergic neurons.

[0502] The presently disclosed subject matter also provides methods of enhancing one or more of survival, engraftment, proliferation, migration, innervation, or differentiation, comprising administering to a cell: (a) a vector encoding a gRNA molecule and a 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 group consisting of GDNF (SEQ ID NO: 10), GBA (SEQ ID NO: 7), and / or SNCA (SEQ ID NO: 8) gene or a functional fragment thereof.

[0503] In one aspect, the disclosure relates to methods for treating or altering cells in a subject (e.g., a human subject or an animal subject), comprising administering to the subject a nucleic acid encoding a Cas protein and first and second guide RNAs (gRNAs) targeting a GDNF (SEQ ID NO: 10), GBA (SEQ ID NO: 7), and / or SNCA (SEQ ID NO: 8) gene of the subject. In some embodiments, the first and second gRNAs target one or more target sequences that encompass or are proximal to 20 nucleic acid target positions, which can be 5', 3', or in the middle of the sequence. The first gRNA can include a targeting domain selected to target the first 20 N (residues 1-20), while the targeting domain of the second gRNA can be selected to target the second 20 N (residues 20-40). The first gRNA can include a targeting domain selected to target the second 20 N (residues 20-40), while the targeting domain of the second gRNA can be selected to target the third 20 N (residues 40-60). The first gRNA can include a targeting domain selected to target the third 20 N (residues 40-60), while the targeting domain of the second gRNA can be selected to target the fourth 20 N (residues 60-80). The first gRNA can include a targeting domain selected to target the second 20 N (residues 20-40), while the targeting domain of the second gRNA can be selected to target the fifth 20 N (residues 80-100).

[0504] The Cas protein can comprise one or more of a nuclear localization signal (NLS) and / or a polyadenylation signal, the Cas protein can be a modified Cas protein (e.g., a Cas9 engineered to alter PAM specificity, improve fidelity, or alter or improve another structural or functional aspect of Cas9). Certain embodiments feature a Cas protein that includes both a C-terminal and an N-terminal NLS. Optionally, the Cas protein is driven by a promoter selected from the group comprising synapsin 1, DAT, VMAT, TH, AADC, a tamoxifen-inducible promoter, a RU486-inducible promoter, or other promoters that allow for temporal control or small molecule control. In various cases, the nucleic acid further comprises first and second inverted terminal repeat sequences (ITRs).

[0505] The embodiments disclosed herein can be further understood by the following numbered paragraphs:

[0506] 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 alter a PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene.

[0507] Paragraph 2. The vector of paragraph 1, wherein the Cas molecule is a Staphylococcus aureus Cas9 molecule.

[0508] Paragraph 3. The vector of paragraph 1, wherein the Cas molecule recognizes a protospacer adjacent motif (PAM) of NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42).

[0509] Paragraph 4. The vector of paragraph 1, wherein the vector is a viral vector.

[0510] Paragraph 5. A composition comprising the vector of paragraph 1.

[0511] Paragraph 6. A genome editing system comprising:

[0512] (a) a gRNA molecule comprising a targeting domain configured to alter a nucleotide sequence set forth in SEQ ID NO: 1-10 or comprising a nucleotide sequence set forth in SEQ ID NO: 1-10; and

[0513] (b) a Cas molecule.

[0514] Clause 7. The genome editing system of clause 6, wherein the genome editing system is configured to alter a PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene.

[0515] Clause 8. The genome editing system of clause 6, wherein the Cas molecule is a Staphylococcus aureus Cas9 molecule.

[0516] Clause 9. The genome editing system of clause 6, wherein the Cas molecule recognizes a PAM of NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42).

[0517] Clause 10. An isolated cell comprising the vector of clause 1.

[0518] Clause 11. An isolated cell comprising the genome editing system of clause 6.

[0519] Clause 12. A method of making a gene edited cell, comprising administering to a cell:

[0520] (a) a vector encoding a gRNA molecule and a Cas protein molecule; or

[0521] (b) a genome editing system comprising a gRNA molecule and a Cas protein molecule;

[0522] wherein the gRNA molecule comprises a targeting domain comprising a portion of the nucleotide sequence set forth in SEQ ID NOs: 1-10 or is configured to alter the nucleotide sequence set forth in SEQ ID NOs: 1-10.

[0523] Clause 13. The method of clause 12, wherein the cell is selected from the group comprising or consisting of: a pluripotent cell and a DA neuronal cell.

[0524] Clause 14. A method of inhibiting degeneration or death of a dopaminergic neuron comprising a mutation in a gene associated with Parkinson’s Disease (PD), the method comprising:

[0525] contacting the neuron with the recombinant gene therapy vector of any one of clauses 1-4;

[0526] wherein upon contact with the recombinant gene therapy vector, the neuron expresses a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, SNCA+ / - and / or GBA protein.

[0527] Clause 15. The method of clause 14, wherein the neuron expresses a reduced amount of alpha-synuclein upon contact with the recombinant gene therapy vector.

[0528] Paragraph 16. The method of any of paragraphs 14-15, wherein the neurons produce and / or release an increased amount of dopamine after contact with the recombinant gene therapy vector.

[0529] Paragraph 17. The method of any of paragraphs 14-16, wherein the neurons express a lower amount of alpha-synuclein protein compared to the amount of alpha-synuclein protein expressed in neurons not contacted with the recombinant gene therapy vector, optionally wherein 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% less than the amount expressed in neurons not contacted with the recombinant gene therapy vector.

[0530] Paragraph 18. The method of any of paragraphs 14-17, wherein the neurons produce and / or release an increased amount of dopamine compared to the amount of dopamine produced and / or released by neurons not contacted with the recombinant gene therapy vector, optionally wherein 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 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater than the amount produced and / or released by neurons not contacted with the recombinant gene therapy vector.

[0531] Paragraph 19. The method of any of paragraphs 14-18, wherein the neurons undergo an increased amount of autophagy compared to the amount of autophagy experienced by neurons not contacted with the recombinant gene therapy vector, optionally wherein 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 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater than the amount experienced by neurons not contacted with the recombinant gene therapy vector.

[0532] Paragraph 20. The method of any of paragraphs 14-19, wherein the neurons are primary tyrosine hydroxylase positive neurons.

[0533] Paragraph 21. A method of treating or inhibiting the onset of Parkinson’s Disease (PD) in a patient having or at risk of PD, the method comprising:

[0534] administering to the subject the recombinant gene therapy vector of any of paragraphs 1-4;

[0535] wherein administration of the recombinant gene therapy vector treats or inhibits the onset of Parkinson’s Disease in the subject.

[0536] Paragraph 22. The method of paragraph 22, wherein the subject is an adult or a child.

[0537] Paragraph 23. The method of any one of paragraphs 21-22, wherein the number of dopaminergic neurons in the subject after the administering step is greater than the number of dopaminergic neurons in the subject before the administering step.

[0538] Paragraph 24. The method of any one of paragraphs 21-23, wherein the level of dopamine in the subject after the administering step is greater than the level of dopamine in the subject before the administering step.

[0539] Paragraph 25. The method of any one of paragraphs 21-24, wherein the number of dopaminergic neurons in the subject treated by the method is increased compared to the number of dopaminergic neurons in a subject not so treated.

[0540] Paragraph 26. The method of any one of paragraphs 21-25, wherein the level of dopamine in the subject treated by the method is increased compared to the level of dopamine in a subject not so treated.

[0541] Paragraph 27. The method of any one of paragraphs 21-26, wherein the level of dopamine in the substantia nigra of the subject treated by the method is increased compared to the level of dopamine in the substantia nigra of a subject not so treated.

[0542] Paragraph 28. The method of any one of paragraphs 21-27, wherein the level of GDNF and / or GBA in the CSF of the subject after the administering step is greater than the level of GDNF and / or GBA in the CSF of the subject before the administering step.

[0543] Paragraph 29. The method of any one of paragraphs 21-28, wherein the Unified Parkinson’s Disease Rating Scale (UPDRS) score of the subject before the administering step is improved compared to the UPDRS score of the subject before the administering step.

[0544] Paragraph 30. The method of any one of paragraphs 21-29, wherein the level of PRKN in the CSF of the subject treated by the method is increased compared to the level of PRKN in the CSF of a subject not so treated.

[0545] Paragraph 31. The method of any one of paragraphs 21-29, wherein the UPDRS score of the subject treated by the method is improved compared to the UPDRS score of a subject not so treated.

[0546] Paragraph 32. The method of any one of paragraphs 21-31, wherein the neurons of the subject express a reduced amount of alpha-synuclein and / or comprise a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector.

[0547] The embodiments disclosed herein can be further understood by the following numbered paragraphs:

[0548] Paragraph 1. An engineered pluripotent cell comprising an exogenous sequence encoding a GDNF gene that has been integrated under transcriptional control of an endogenous gene promoter.

[0549] Paragraph 2. An engineered DA neuronal cell comprising an exogenous sequence encoding a GDNF gene that has been integrated under transcriptional control of an endogenous gene promoter.

[0550] Paragraph 3. The engineered pluripotent cell of paragraph 1 having the genotype SNCA+ / -.

[0551] Paragraph 4. The engineered DA neuronal cell of paragraph 2 having the genotype SNCA+ / -.

[0552] Methods

[0553] Methods for making engineered cells

[0554] In some embodiments, a method of making an engineered cell is disclosed, the method comprising contacting a pluripotent cell with a gene editing system comprising: a Cas protein or a polynucleotide encoding a Cas protein; a guide RNA (gRNA); and a repair template comprising 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.

[0555] In some embodiments, the pluripotent cell is in vitro. In some embodiments, the engineered pluripotent cell is differentiated into a DA neuronal cell in vitro to form an engineered DA neuronal cell. In some embodiments, the engineered DA neuronal cell is translated into a patient. In some embodiments, the engineered DA neuronal cell is capable of enhancing viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of the administered engineered DA neuronal cell compared to a wild-type neuronal cell. In some embodiments, the administered engineered DA neuronal cell differentiates into a neuronal mature cell type in vivo and the administered engineered DA neuronal cell enhances function of the neuronal mature cell type compared to a wild-type neuronal mature cell. In some embodiments, the administered engineered DA neuronal cell enhances function of an endogenous (non-engineered) neuronal mature cell type in the patient.

[0556] In some embodiments, the pluripotent cell and / or subject to be genetically engineered comprises a mutation in the PARK2 gene, the PINK1 gene, the LRRK2 gene, the c-Rel gene, the ATG7 gene, VMAT2, GBA, SNCA, MAPT, and / or GDNF gene. In some embodiments, the pluripotent cell and / or subject to be genetically engineered comprises a mutation in one of SEQ ID NOs: 1-10, or a combination thereof.

[0557] Disclosed herein are methods for making an engineered cell, comprising: introducing into a cell a guide polynucleotide comprising a spacer that is complementary to a target nucleic acid in a genomic region of the cell; a nuclease guided by the guide polynucleotide; and a polynucleotide encoding an exogenous GDNF, GBA, or SNCA; site-specifically cleaving the target nucleic acid within the cell by the nuclease guided by the guide polynucleotide; and inserting the polynucleotide encoding the exogenous GDNF, GBA, and / or SNCA into the genomic region of the cell at the cleavage site. The nuclease can be Cas9. In some embodiments, the guide polynucleotide can be a single guide polynucleotide. The guide polynucleotide can be RNA. The target nucleic acid can be DNA. The spacer can be between 10-30 nucleotides in length. The nuclease can create a double-stranded break in the target nucleic acid. The nuclease can create a single-stranded break in the target nucleic acid.

[0558] In some embodiments, the guide polynucleotide can be introduced into the cell by electroporation. The guide nucleic acid can be introduced into the cell by nucleofection. The nuclease can also be introduced into the cell by a delivery vehicle. The polynucleotide encoding the exogenous GDNF, GBA, and / or SNCA can further comprise a promoter sequence. The promoter sequence can be selected from one of SEQ ID NOs: 21-40 or 43. The exogenous GDNF, GBA, and / or SNCA can be inserted by homologous recombination. The guide polynucleotide and the nuclease can form a ribonucleoprotein complex.

[0559] Cleaving the target nucleic acid can remove a genomic nucleic acid sequence that is replaced by the polynucleotide encoding the exogenous GDNF, GBA, and / or SNCA. The polynucleotide encoding the exogenous GDNF, GBA, and / or SNCA can further comprise a first recombination arm and a second recombination arm. The first recombination arm can comprise a first sequence that is identical to a first portion of the target nucleic acid, and the second recombination arm can comprise a second sequence that is identical to a second portion of the target nucleic acid. In some embodiments, the first recombination arm can comprise a first sequence that is identical to a first portion adjacent to the target nucleic acid, and the second recombination arm can comprise a second sequence that is identical to a second portion adjacent to the target nucleic acid. The target nucleic acid can be within a gene. The gene can be selected from GDNF, GBA, and / or SNCA.

[0560] In some embodiments, insertion of an exogenous GDNF, GBA, and / or SNCA sequence at a cleavage site can result in disruption of a gene. The target nucleic acid can be within an intergenic site. The exogenous GDNF, GBA, and / or SNCA can be expressed in a cell. The engineered cell can be introduced into an organism. The engineered cell can be expanded ex vivo.

[0561] Disclosed herein are methods for efficient target gene disruption in DA neuronal cells, comprising contacting a pluripotent cell with a Cas protein nuclease and a guide RNA, wherein the guide RNA contains a region of 17 to 22 nucleotides that is substantially complementary to a region in a target gene; cleaving the target gene, wherein the target gene can be GDNF, GBA, and / or SNCA, and wherein when a population of pluripotent cells is contacted with a Cas protein nuclease and a guide RNA, exogenous knockout events occur in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the pluripotent cells.

[0562] In some embodiments, the method comprises one or more cells, and the one or more cells are pluripotent cells, DA neuronal cells, neurons, or any combination thereof. In some embodiments, the method comprises a first nucleic acid, and the first nucleic acid is DNA, RNA, or a hybrid thereof. In some embodiments, the method comprises a first nucleic acid, and the first nucleic acid is single-stranded or double-stranded. In some embodiments, the method comprises a second nucleic acid, and the second nucleic acid is DNA, RNA, or a hybrid thereof. In some embodiments, the method comprises a second nucleic acid, and the second nucleic acid is single-stranded or double-stranded. In some embodiments, the method comprises introducing a first nucleic acid, and introducing the first nucleic acid comprises non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method comprises viral transduction, and the viral transduction comprises adeno-associated virus. In some embodiments, the method comprises introducing a second nucleic acid, and introducing the second nucleic acid comprises non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection.

[0563] In some embodiments, the method comprises a double strand break, and generating the double strand break comprises CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL. In some embodiments, the method comprises a double strand break, and generating the double strand break comprises CRISPR. In some embodiments, the method comprises 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 comprises a second nucleic acid, and the second nucleic acid comprises a recombination arm, and wherein the second gene encoding an engineered GDNF, GBA, and / or SNCA gene is flanked by the recombination arm. In some embodiments of the methods of the disclosure, an increase in the isogenicity between the recombination arm and the at least one endogenous GDNF, GBA, and / or SNCA gene corresponds to an increase in the insertion efficiency of the second gene. In some embodiments, the method comprises inserting a second gene, and the insertion efficiency of the second gene is measured using fluorescent expression cell sorting. In some embodiments, the method comprises introducing a second nucleic acid, and introducing the second nucleic acid comprises non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method comprises inserting a second gene, and inserting the second gene encoding an engineered GDNF, GBA, and / or SNCA gene comprises homology directed repair (HDR). In some embodiments, the method comprises inserting a second gene, and the insertion of the second gene is assisted by a homologous recombination (HR) enhancer. In some embodiments, the method comprises an enhancer, and the enhancer is derived from a viral protein. In some embodiments, the method comprises an HR enhancer, and the HR enhancer is selected from the group comprising or consisting of E4orf6, Elb55K, Elb55K-H354, Elb55K-H373A, Scr7, L755507, or any combination thereof. In some embodiments, the method comprises an HR enhancer, and the HR enhancer is a chemical inhibitor. In some embodiments, the method comprises an HR enhancer, and the HR enhancer inhibits ligase IV. In some embodiments, the method comprises a reduction in cellular toxicity, and the cellular toxicity comprises at least one of the following: DNA cleavage, cell death, apoptosis, nuclear condensation, cell lysis, necrosis, altered cell motility, altered cell stiffness, altered cytoplasmic protein expression, altered membrane protein expression, swelling, loss of membrane integrity, cessation of metabolic activity, low active metabolism, high active metabolism, increased reactive oxygen species, cytoplasmic contraction, or any combination thereof.In some embodiments, the method comprises measuring viability, and the viability is measured using at least one of fluorescence activated cell sorting, trypan blue exclusion, CD4+ cell surface markers, CD8+ cell surface markers, telomere length, or any combination thereof. In some embodiments, the method comprises a subject, and the subject is a human subject.

[0564] Methods of making a therapeutically effective composition

[0565] In one embodiment, a method of making a therapeutically effective composition comprising one or more cells is disclosed. In some embodiments, the method comprises gene editing, and the gene editing comprises introducing a first nucleic acid into the one or more cells. In some embodiments, the method comprises a first nucleic acid, and the first nucleic acid comprises a first gene encoding a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF protein. In some embodiments, the method comprises gene editing, and the gene editing comprises introducing a second nucleic acid into the one or more cells. In some embodiments, the method comprises a second nucleic acid, and the second nucleic acid comprises a second gene encoding a PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF protein, wherein the second gene is different from the first gene.

[0566] A method of treating or inhibiting the onset of Parkinson’s disease (PD) in a subject having or at risk of having PD is disclosed, comprising:

[0567] administering DA neuronal cells to a subject, wherein the DA neuronal cells comprise a recombinant genetic 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 the DA neuronal cells comprising the recombinant genetic vector treats or inhibits the onset of Parkinson’s disease in the subject.

[0568] Methods of identifying a safe harbor locus

[0569] Methods of identifying safe harbor loci are disclosed. Methods of identifying safe harbor loci suitable for editing PSCs and expression in post-mitotic dopaminergic neurons comprise, consist of, or consist essentially of: a. generating prioritized genomic safe harbor sites from clinically compliant PSC lines and / or dopaminergic neuronal progenitor cells using scATAC-seq data; b. comparing data from step a to published ATAC-seq data from dopaminergic neurons isolated from human healthy and Parkinson’s brains; c. defining shared regions of chromatin accessibility between the data in a and the comparison in step b, thereby generating a prioritized catalog of GSH sites suitable for editing in PSCs and expression in post-mitotic dopaminergic neurons. In some embodiments, the prioritized catalog of GSH sites is further prioritized based on GSH sites that are distal to genes, regulatory sequences, telomeres, and centromeres to prevent transgene interference with gene expression, division, and function. In some embodiments, GSH sites are selected based on highest efficiency of transgene insertion, long-lasting expression after extended culture, and / or lack of oncogenic expression. In some embodiments, GSH sites are selected based on peak size and / or distribution data generated with processed ATAC-seq data.

[0570] Disclosed is a method of identifying a safe harbor locus suitable for editing PSCs and expressing in post-mitotic dopaminergic neurons, the method comprising, consisting of, or consisting essentially of: a. generating prioritized genomic safe harbor sites from clinical compliant PSC lines and / or dopaminergic neuronal progenitor cells using scATAC-seq data; b. comparing data from step a to published ATAC-seq data from dopaminergic neurons isolated from human healthy and Parkinson’s brains; c. defining regions of shared chromatin accessibility between the data in a and the comparison in step b; and d. selecting a safe harbor locus based on a threshold parameter; wherein the safe harbor locus is selected for insertion of at least one sequence encoding a transgene within the cell. In some embodiments, the threshold parameter is based on peak size and / or distribution data generated with processed ATAC-seq data. In some embodiments, the threshold parameter is based on peak size data generated with processed ATAC-seq data, distribution data generated with processed ATAC-seq data, highest efficiency of transgene insertion, persistent expression after extended culture, and / or lack of oncogenic gene expression. In some embodiments, defining chromatin accessibility is based on (1) PSCs to optimize editing efficiency in step a, (2) DA neuronal cells to optimize editing efficiency in step a, and (3) post-mitotic dopaminergic neurons from human brain samples. In some embodiments, defining chromatin accessibility is based on (1) PSCs to optimize editing efficiency in step a, (2) DA neuronal cells to optimize editing efficiency in step a, (3) post-mitotic dopaminergic neurons from human brain samples, and (4) neuronal mature cell type cells.

[0571] In some embodiments, the method of targeting a transgene (e.g., GDNF) into a safe harbor locus in PSCs for expression in post-mitotic dopaminergic neurons further comprises selecting sgRNAs with the highest on-target activity and lowest off-target activity, and empirically defining the best cutting sgRNAs.

[0572] In some embodiments, identifying a safe harbor locus in PSCs for expression in post-mitotic dopaminergic neurons further comprises further defining chromatin accessibility based on ATAC-seq peak data in the first cell type (PSC, DA neuron, and / or neuron mature cell type) compared to ATAC-seq peak data in a second, different cell type (PSC cell, DA neuron cell, neuron mature cell type, and / or HEK cell). In some embodiments, chromatin accessibility is based on ATAC-seq peak data comparing to validated transgene insertion, and / or alignment between ATAC-seq peaks and sgRNA locations. In some embodiments, chromatin accessibility is based on ATAC-seq peak data comparing scATAC-seq data from substantia nigra (SN) cells from healthy controls and Parkinson’s disease patients. In some embodiments, chromatin accessibility is based on ATAC-seq peak data comparing scATAC-seq data from substantia nigra (SN) cells from healthy controls and Parkinson’s disease patients compared to published ATAC-seq from human iPSCs and mature dopaminergic neurons. In some embodiments, chromatin accessibility is based on ATAC-seq peak data comparing scATAC-seq data from dopaminergic cells from healthy controls and Parkinson’s disease patients. In some embodiments, chromatin accessibility is based on ATAC-seq peak data comparing scATAC-seq data from substantia nigra (SN) cells from healthy controls and Parkinson’s disease patients compared to published ATAC-seq 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 “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 “open” in Parkinson’s disease patients. In some embodiments, chromatin accessibility is based on any combination of factors discussed in this paragraph. In some embodiments, transgene insertion (e.g., GDNF) has no effect on differentiation capacity, i.e., DA neuron cells can differentiate (in vivo and in vitro) into neuron mature cell types.

[0573] Disclosed is a method of identifying a safe harbor locus, the method comprising, consisting of, or consisting essentially of: a. identifying genes or non-coding regions in a chromosome that are above a threshold level of chromatin accessibility; b. generating a model that correlates the genes or non-coding regions from step (a) with published ATAC-seq data from dopaminergic neurons isolated from human healthy and Parkinsonian brains with respect to the chromosome; and c. selecting a safe harbor locus based on a threshold parameter; wherein the safe harbor locus is selected for insertion of at least one sequence encoding a transgene within a cell. In some embodiments, the threshold parameter is based on peak size data generated with processed ATAC-seq data, distribution data generated with processed ATAC-seq data, highest efficiency of transgene insertion, durable expression after prolonged culture, lack of oncogene expression, stable expression of transgene, gene knockout that confers a benefit to cell function, no known function within a cell, stable transgene expression in vitro, negligible off-target cleavage as detected by iGuide-Seq or CRISPR-Seq, less off-target cleavage relative to other loci as detected by iGuide-Seq or CRISPR-Seq, negligible transgene-independent cellular toxicity, negligible transgene-independent cytokine expression, negligible transgene-independent chimeric antigen receptor expression, negligible de-regulation or silencing of nearby genes, wherein chromatin accessibility is measured using transposase accessible chromatin sequencing assay (ATAC-seq). In some embodiments, chromatin accessibility is based on (1) PSCs to optimize editing efficiency in step a, and / or (2) DA neuronal cells to optimize editing efficiency in step a, and (3) post-mitotic dopaminergic neurons from human brain samples. In some embodiments, defining chromatin accessibility is based on (1) PSCs to optimize editing efficiency in step a, (2) DA neuronal cells to optimize editing efficiency in step a, (3) post-mitotic dopaminergic neurons from human brain samples to the extent that the engineered cells are able to mature into a neuronal mature cell type to facilitate endogenous neuron survival, and (4) neuronal mature cell type cells.

[0574] Disclosed is an ex vivo method of obtaining an engineered cell or population thereof comprising: a. obtaining a cell; b. genetically modifying the cell by inserting at least one sequence encoding a transgene within a safe harbor locus, wherein the safe harbor locus is selected from any one of the target loci in Table 1. Wherein the genetic modification in step (b) comprises contacting the cell with one or more guide ribonucleic acid (gRNA), at least one sequence, and one or more Cas9 endonuclease, wherein the one or more gRNA and Cas9 endonuclease facilitate insertion of the at least one sequence into the chromosomal DNA within the safe harbor locus. Wherein the at least one sequence comprises an exogenous promoter, and the exogenous promoter is operably linked to the transgene. Wherein the transgene comprises, consists of, or consists essentially 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 functional variants or fragments thereof.

[0575] Gene targeting insertion in pluripotent stem cells (PSCs)

[0576] Methods are provided for gene targeting insertion in PSCs to introduce an exogenous coding sequence under the control of an endogenous promoter, particularly an endogenous promoter for a gene that is specifically expressed in cells of a particular neuronal lineage or at a particular stage of differentiation, preferably at a late stage of differentiation. In some embodiments, the PSCs can be transduced with a polynucleotide vector (donor template) such as an AAV vector during ex vivo therapy as discussed herein, while expressing a sequence-specific nuclease reagent to facilitate insertion of the coding sequence at the selected locus. In some embodiments, the PSCs can be engineered using CRISPR, TALEN, transposition-based, ZFN, meganucleases, or Mega-TALs to generate a double-strand break that is repaired by insertion of a gene encoding an engineered GDNF, GBA, and / or SNCA gene. The resulting engineered PSCs can then be differentiated into engineered DA neuronal cells and implanted into a patient in need thereof for long-term in vivo production of the exogenous coding sequence. Depending on the activity of the selected endogenous promoter, the coding sequence can be selectively expressed in certain lineages or in response to the local environment of the DA neuronal cells in vivo, thereby providing treatment for Parkinson's disease and other and secondary parkinsonian disorders.

[0577] In some embodiments, the exogenous coding sequence is placed under the control of a gene promoter whose transcriptional activity is specifically induced in the cell (i.e., a cell of a mature neuronal cell type) after implant function has been established. In some embodiments, the exogenous coding sequence is placed under the control of a gene promoter whose transcriptional activity is specifically induced in the cell after cell transplantation has occurred. In some embodiments, the exogenous coding sequence is placed under the control of a gene promoter whose transcriptional activity is not specifically induced in a cell differentiated in vitro. In some embodiments, the exogenous coding sequence is placed under the control of a gene promoter whose transcriptional activity is not specifically induced in a cell expanded in vitro. In some embodiments, the exogenous coding sequence is placed under the control of a gene promoter whose transcriptional activity is specifically induced in the cell only after implant function has been established.

[0578] It is disclosed to introduce an exogenous sequence encoding GDNF, GBA, SCNA + / - or components thereof into PSCs, preferably under the transcriptional control of a gene promoter that is not expressed in pluripotent cells or differentiated cells or DA neuronal cells. 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 neural cells produced from 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.

[0579] In some embodiments, an exogenous sequence encoding GDNF or components thereof is introduced into ESCs under the transcriptional control of a gene described as specifically expressed in, preferably only in, mature neuronal cell types. In some embodiments, the GDNF gene is under the transcriptional control of one of SEQ ID NOs: 21-40 or 43.

[0580] In some embodiments, the PSCs comprise an exogenous coding sequence that is expressed only in a selected neural lineage. More broadly, methods are disclosed for engineering PSCs by gene-targeted insertion of an exogenous coding sequence to be selectively expressed in a neuronal mature cell derived from the PSC. As an embodiment, the neuronal mature cell produced from the engineered PSC expresses the exogenous coding sequence in response to a selected environmental factor or in vivo stimulus to improve their therapeutic potential. More broadly, methods are disclosed for engineering PSCs by gene-targeted insertion of an exogenous coding sequence to be selectively expressed in an in vivo environment in which the neuronal mature cell resides. As an embodiment, the engineered cell derived from the engineered PSC expresses the exogenous coding sequence in response to a selected environmental factor or in vivo stimulus to improve their therapeutic potential for the neuronal mature cell.

[0581] Insertion of a targeting sequence in a DA neuronal cell in combination with inactivation of an endogenous genomic sequence

[0582] In some embodiments, the inactivation of a gene at a locus in a DA neuronal cell is achieved by integration of an exogenous coding sequence at the locus, expression of which improves the therapeutic potential of the engineered cell.

[0583] In some embodiments, the insertion of a coding sequence has the effect of reducing or preventing expression of the gene involved.

[0584] Expansion of DA neuronal cells

[0585] Engineered pluripotent cells and / or DA neuronal cells can be expanded, whether prior to or after genetic modification. Pluripotent cells and / or DA neuronal cells can be expanded in vitro or in vivo. In some embodiments, engineered pluripotent cells and / or DA neuronal cells do not express the exogenous gene during expansion. In some embodiments, engineered pluripotent cells and / or DA neuronal cells do express the exogenous gene during expansion.

[0586] Gene editing

[0587] In some embodiments, methods are disclosed comprising the steps of:

[0588] - providing a population of pluripotent cells;

[0589] - introducing into a proportion of the pluripotent cells:

[0590] i) at least one nucleic acid comprising an exogenous nucleotide or polynucleotide sequence, thereby producing an engineered pluripotent cell, wherein the exogenous nucleotide or polynucleotide is integrated at a selected endogenous locus to encode at least one molecule for improving the therapeutic potential of a population of DA neuronal cells derived from the engineered pluripotent cell;

[0591] ii) at least one sequence-specific reagent that specifically targets a selected endogenous locus, wherein the exogenous nucleotide or polynucleotide sequence is inserted into the endogenous locus by targeted gene integration such that the exogenous nucleotide or polynucleotide sequence forms an exogenous coding sequence under the transcriptional control of an endogenous promoter present at the locus.

[0592] In some embodiments of the method, the sequence-specific reagent is a nuclease, and the targeted gene integration operates on the pluripotent cell by homologous recombination or non-homologous end joining (NHEJ). In some embodiments, NHEJ can be inhibited in the cell. Inhibiting NHEJ in the cell can include inhibiting ligase IV. Inhibiting NHEJ in the cell can also include introducing a homologous recombination (HR) enhancer. The enhancer can be derived from a viral protein. The enhancer can be ElB55K, E4ORF6, Scr7, or L755507. Inhibiting NHEJ in the cell can facilitate insertion of the polynucleotide encoding the exogenous GDNF, GBA, and / or SNCA at the cleavage site by homologous recombination.

[0593] In some embodiments, the endogenous promoter is selected to be inactive during in vitro cell differentiation and is preferably upregulated after implant function has been established in vivo. In some embodiments, the endogenous promoter is selected to be inactive during in vitro cell differentiation and is preferably upregulated after the cells are transplanted into a subject.

[0594] The method can be further understood by the following numbered paragraphs:

[0595] Paragraph 1. A method of treating Parkinson’s disease and other and secondary parkinsonian conditions in a human subject, the method comprising: administering to the human subject an effective amount of a pharmaceutical composition comprising (i) a population of genetically modified human DA neuronal cells comprising a genomic disruption in an endogenous gene that suppresses or eliminates expression of a protein encoded by the gene; and (ii) a pharmaceutically acceptable carrier or excipient.

[0596] Paragraph 2. The method of paragraph 1, wherein the gene is a GDNF, GBA, and / or SNCA gene.

[0597] Paragraph 3. The method of paragraph 1, wherein the DA neuronal cells are autologous to the human subject.

[0598] Paragraph 4. The method of paragraph 1, wherein the genomic disruption is a nucleotide insertion or deletion in the gene.

[0599] Paragraph 5. The method of paragraph 1, wherein the genetically modified human DA neuronal cells comprise an exogenous GDNF, GBA, and / or SNCA gene.

[0600] Paragraph 7. The method of paragraph 1, wherein the genetically modified DA neuronal cell is derived from a genetically modified pluripotent stem cell.

[0601] Gene editing for SNCA hemizygous invalidation

[0602] In one embodiment, a method for adoptive cell therapy or prevention is provided, comprising administering to a subject having an alpha-synuclein accumulation condition or at risk of having an alpha-synuclein accumulation condition, a DA neuronal cell modified for SNCA. In some embodiments, the DA neuronal cell modified for SNCA is autologous. In some embodiments, the DA neuronal cell modified for SNCA is allogeneic.

[0603] In some embodiments, the DA neuronal cell modified for SNCA is a DA neuronal cell genetically modified to have reduced SNCA expression. In some embodiments, the DA neuronal cell modified for SNCA is a DA neuronal cell genetically modified to have reduced SNCA expression and increased GBA expression.

[0604] In some embodiments, the DA neuronal cell modified for SNCA is derived from a pluripotent cell genetically modified to have reduced SNCA expression, for example by gene editing with a programmable nuclease or by gene silencing (RNA interference). In some embodiments, the genetically modified DA neuronal cell modified for SNCA is a SNCA − / − In some embodiments, the genetically modified DA neuronal cell modified for SNCA is a SNCA + / − In some embodiments, the genetically modified DA neuronal cell modified for SNCA is a SNCA + / + .

[0605] DA neuronal cells modified for SNCA can be further understood by the numbered paragraphs below:

[0606] Paragraph 1. A method of obtaining a human DA neuronal cell, comprising differentiating a human pluripotent stem cell into a human DA neuronal cell, wherein the human pluripotent cell is a genetically modified cell in which one or both SNCA alleles have been rendered non-expressing by genetic modification.

[0607] Paragraph 2. The method of paragraph 1, wherein both SNCA alleles have been rendered non-expressing by genetic modification.

[0608] Paragraph 3. The method of paragraph 1, wherein the human pluripotent stem cell is an induced pluripotent stem cell.

[0609] Paragraph 4. The method of paragraph 3, wherein both SNCA alleles have been rendered non-expressing by genetic modification.

[0610] Paragraph 5. The method of paragraph 1, comprising expanding the human DA neuronal cells.

[0611] Paragraph 6. The method of paragraph 1, wherein the human pluripotent cells are genetically modified using CRISPR / Cas protein gene editing.

[0612] Paragraph 7. A population of human cells comprising human DA neuronal cells, wherein the human DA neuronal cells are genetically modified human DA neuronal cells, wherein one or both SNCA alleles have been rendered non-expressing by genetic modification.

[0613] Cytotoxicity

[0614] It can be further disclosed herein to include introducing a modifying agent to the cells to reduce cytotoxicity. The modifying agent can be the pan-Caspase inhibitor Z-VADFMK and / or BX795. The cells can be pluripotent cells, DA neuronal cells, or neural cells. The cells can be mammalian cells. The cells can be human cells.

[0615] CIS and IRES

[0616] In some embodiments, methods are disclosed comprising gene editing of pluripotent cells (or in some embodiments DA neuronal cells) to maintain expression of the native gene with integrated gene transcription under the control of the endogenous promoter by using a CIS regulatory element (e.g., 2A CIS acting hydrolyase element) or internal ribosome entry site (IRES) in the donor template.

[0617] Upregulation of GDNF after DA neuronal cell differentiation is complete

[0618] In some embodiments, methods are disclosed comprising generating a double-strand break at a highly transcribed locus once the function of the implant has been established, which is performed by expressing a sequence-specific nuclease reagent, which is, as non-limiting examples, e.g., a TALEN, ZFN, or RNA-guided endonuclease, in the presence of a DNA repair substrate, preferably in the presence of a DNA repair substrate provided into an AAVS1 -based vector. The DNA donor template typically comprises two homology arms, which are embedded into a unique or multiple open reading frames and regulatory genetic elements (stop codon and polyA sequence).

[0619] Methods of expressing GDNF at a selected genetic locus that is upregulated after completion of differentiation, engraftment, proliferation, migration, and innervation of DA neuronal cells in vivo, i.e., upregulated once engraftment function has been established or once the administered engineered DA neuronal cells have differentiated into a neuronal mature cell type, are disclosed. An exogenous sequence encoding a GDNF gene or functional fragment thereof and an endogenous gene-encoding sequence can be co-transcribed, e.g., separated by a CIS regulatory element (e.g., a 2A CIS-acting hydrolyase element) or by an internal ribosome entry site (IRES). For example, an exogenous sequence encoding a GDNF gene or functional fragment thereof can be placed under transcriptional control of the promoter of an endogenous gene expressed by the engraftment microenvironment.

[0620] GBA upregulation after completion of DA neuronal cell engraftment

[0621] In some embodiments, methods are disclosed that include generating a double-strand break at a highly transcribed locus after engraftment of engineered cells, by expressing a sequence-specific nuclease reagent, as a non-limiting example, e.g., a TALEN, ZFN, or RNA-guided endonuclease, in the presence of a DNA repair substrate, preferably provided in an AAVS1 -based vector. The DNA donor template typically includes two homology arms that flank a unique or multiple open reading frames and regulatory genetic elements (stop codon and polyA sequence).

[0622] Methods of expressing GBA at a selected genetic locus that is not upregulated after completion of DA neuronal cell engraftment, but is upregulated after DA neuronal cell engraftment, i.e., upregulated before engraftment function has been established or before the administered DA neuronal cells have differentiated into a neuronal mature cell type, are disclosed. An exogenous sequence encoding a GBA gene or functional fragment thereof and an endogenous gene-encoding sequence can be co-transcribed, e.g., separated by a CIS regulatory element (e.g., a 2A CIS-acting hydrolyase element) or by an internal ribosome entry site (IRES). For example, an exogenous sequence encoding a GBA gene or functional fragment thereof can be placed under transcriptional control of the promoter of an endogenous gene expressed by the engraftment, or they can be a ubiquitous promoter.

[0623] A new framework for identifying candidate genomic safe harbor ("GSH") is disclosed in Aznauryan et al., Cell Reports Methods, 2, 100154 (2022). Candidate GSHs are selected based on ATAC-seq data showing favorable chromatin accessibility profiles in DA neurons. Fullard et al., Genome Research, 28: 1243-1252 (2021) show candidate GSH loci with evidence of accessible chromatin in the human striatum (nucleus accumbens and putamen), and Corces et al., Nature Genetics, 52: 1158-1168 (2020).

[0624] Contacting a neural cell with an engineered DA neuron cell in vivo

[0625] In some embodiments, a method of promoting viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of an administered engineered DA neuron cell is disclosed, the method comprising contacting a neuron with a gene editing system comprising: a Cas protein or a polynucleotide encoding a Cas protein; a guide RNA (gRNA); 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; wherein the gene editing system is capable of enhancing viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous neurons, or function of an administered engineered DA neuron cell compared to a wild-type neuron cell. In some embodiments, the neuron is in a patient when it is contacted with the engineered DA neuron cell.

[0626] A method of engrafting, proliferating, or promoting viability, migration, innervation, differentiation, or function of an administered engineered DA neuron cell is disclosed, the method comprising a recombinant gene vector comprising a polynucleotide encoding a wild-type PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / - and / or GDNF gene or a functional variant or fragment thereof; wherein the polynucleotide is operably linked to a eukaryotic active promoter; and wherein the DA neuron cell transduced with the recombinant gene expresses the wild-type protein or a functional variant or fragment thereof upon engraftment into a host / patient. In some embodiments, the DA neuron cell transduced with the recombinant gene expresses the wild-type protein or a functional variant or fragment thereof upon maturation in the host / patient.

[0627] Non-viral introduction of exogenous genes into cells

[0628] Also disclosed herein are methods for making an engineered cell comprising a) introducing one or more polynucleic acids comprising at least one exogenous GDNF, GBA, and / or SNCA sequence into a cell in a non-viral manner; and b) contacting the at least one exogenous GDNF, GBA, and / or SNCA exogenous sequence with a double-stranded break region comprising at least one gene. The at least one gene can be GDNF, GBA, and / or SNCA. The double-stranded break region can be repaired by insertion of the at least one exogenous GDNF, GBA, and / or SNCA sequence. The insertion of the at least one exogenous GDNF, GBA, and / or SNCA sequence can comprise a disruption of the at least one gene.

[0629] In some embodiments, the gene introduction can comprise non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. The polynucleic acid can be co-delivered with at least one modifying agent that alters the cell's response to the polynucleic acid. The at least one modifying agent can reduce cellular toxicity. The modifying agent can comprise abPanCaspase inhibitor Z-VAD-FMK or BX795.

[0630] Introduction of exogenous genes into cells via double-stranded break regions

[0631] Also disclosed herein are methods for making an engineered cell comprising introducing a double-stranded break region into a cell. In some embodiments, the double-stranded break region can be generated by CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL. In some embodiments, the double-stranded break region can be generated by CRISPR. In some embodiments, the CRISPR can be multiplexed. In some embodiments, the multiplexing can be by adding at least 2 guide RNAs. The GDNF, GBA, and / or SNCA exogenous sequence can be inserted near the double-stranded break region.

[0632] Introduction of exogenous genes into cells via reverse transcriptases.

[0633] In some embodiments, the cell to be engineered can be contacted with a reverse transcriptase (RT). In some embodiments, the cell can be contacted with a primer complementary to the polynucleic acid. In some embodiments, the RT transcribes the mRNA into a first ssDNA template. In some embodiments, the RT transcribes the first ssDNA template into a second dsDNA template. In some embodiments, the transcription can be performed in situ. The ssDNA or dsDNA can comprise at least one exogenous GDNF sequence. In some embodiments, the presence of the RT can be determined using a primer sequence. The reverse transcriptase (RT) reporter forward primer can be determined by one of ordinary skill in the art by known methods. The reverse transcriptase (RT) reporter forward primer can be determined by being capable of (selectively) binding to a portion of one of SEQ ID NOs: 1-10. In some embodiments, the forward primer is 10 nucleic acids long and can bind to the first 10 N (residues 1-10), the second 10 N (residues 10-20), the third 10 N (residues 20-30), the fourth 10 N (residues 30-50), or the fifth 10 N (residues 50-60) of the amino acid sequence set forth in SEQ ID NOs: 1-10.

[0634] Homology directed repair

[0635] Also disclosed herein are methods for facilitating homology directed repair (HDR) comprising a) introducing mRNA, reverse transcriptase (RT), an enhancer, and a primer into a cell in a non-viral manner; b) reverse transcribing the mRNA into one or more copies of a polynucleic acid; and c) facilitating HDR between the genome of the cell and the genome of the polynucleic acid. In some embodiments, the method can comprise generating a double strand break. In some embodiments, the double strand break can be performed by CRISPR, TALEN, transposon-based, ZFN, meganucleases, and meganucleases-TAL. In some embodiments, the double strand break can be performed by CRISPR. In some embodiments, the HDR of c) repairs the double strand break. In some embodiments, the CRISPR can be multiplexed with at least two (2) guide RNAs. In some embodiments, the polynucleic acid can be DNA. In some embodiments, the polynucleic acid can be cDNA. In some embodiments, the polynucleic acid can be single stranded.

[0636] In some embodiments, the RT transcribes the mRNA into a first ssDNA template. In some embodiments, the polynucleic acid can be double stranded. In some embodiments, the RT transcribes the mRNA in situ into a second dsDNA template. The mRNA or polynucleic acid can comprise at least one GDNF sequence. In some embodiments, the GDNF sequence comprises at least two flanking recombination arms having sequences complementary to a genomic region. In some embodiments, the GDNF sequence is useful in the HDR of c). In some embodiments, the GDNF sequence is useful in the HDR of c) and further comprises binding of the recombination arms to a complementary portion of the cell genome. In some embodiments, the GDNF sequence is useful in the HDR of c) and further comprises binding of the recombination arms to a complementary portion of the cell genome and further comprises insertion of the GDNF sequence. In some embodiments, the HDR between the genome of the cell and the genome of the polynucleic acid disrupts one or more genes. The one or more genes can comprise XX. In some embodiments, the one or more genes comprise GDNF. In some embodiments, the HDR between the cell genome and the polynucleic acid genome can be aided by one or more homologous recombination (HR) enhancers.

[0637] Targeted homologous recombination

[0638] In some embodiments, an exogenous sequence is introduced into an endogenous chromosomal DNA by targeted homologous recombination. Thus, the exogenous nucleic acid introduced into the pluripotent cell comprises at least one coding sequence, and a sequence that can hybridize to an endogenous chromosomal sequence under physiological conditions. Typically, such homologous sequences show at least 70%, preferably 80% and more preferably 90% sequence identity with the endogenous gene sequence located at the insertion locus. These homologous sequences can flank the coding sequence to improve the precision of recombination, as has been taught, for example, in US 6,528,313, which is incorporated by reference in its entirety.

[0639] Using available software and online genomic databases, one can design a vector to include a coding sequence in such a way that the sequence is introduced at the precise locus under the transcriptional control of at least one endogenous promoter, which is the promoter of the endogenous gene. Then, the exogenous coding sequence is preferably inserted "in-frame" with the endogenous gene. The sequence resulting from integration of the exogenous polynucleotide sequence can encode many different types of proteins, including fusion proteins, tagged proteins, or mutant proteins. Fusion proteins allow the addition of new functional domains to the proteins expressed in the cell, such as dimerization domains that can be used to turn on or off protein activity, such as the caspase-9 switch. Tagged proteins can be advantageous for detecting engineered DA neuronal cells and following patients treated with the cells. Introducing mutations into proteins can confer resistance to drugs, as described further below.

[0640] rare-cutting endonuclease

[0641] Preferably, the sequence-specific reagent used in the method is a rare-cutting endonuclease known to the person skilled in the art. Typically, integration of the manipulation of the targeted gene in pluripotent cells is by homologous recombination or NHEJ. Preferably, the specific endonuclease reagent is selected from the group consisting of RNA or DNA guided endonucleases such as Cas9 or Cpfl, RNA or DNA guides, TAL-nucleases, zinc finger nucleases, homing endonucleases or any combination thereof.

[0642] TALE-nucleases

[0643] In some embodiments, the endonuclease reagent is a nucleic acid encoding a "engineered" or "programmable" rare-cutting endonuclease such as a homing endonuclease as described for example by Arnould S. et al. (WO2004067736), a zinc finger nuclease as described for example by Urnov F. et al. (Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651), a TALE-nuclease as described for example 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 MegaTAL nuclease as described for example by Boissel et al. (MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research 42 (4):2591-2601).

[0644] In some embodiments, the endonuclease reagent is a RNA guide used in combination with a RNA-guided endonuclease such as Cas9 or Cpfl, especially according to the teachings of 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.

[0645] In some embodiments, the endonuclease reagent is transiently expressed into the cell, meaning that the reagent is not considered to integrate into the genome or persist for long periods of time, for example RNA, more particularly mRNA, protein or complex of protein and nucleic acid (e.g. ribonucleoprotein) cases.

[0646] Typically, 80% of the endonuclease reagent is degraded 30 hours, preferably 24 hours, more preferably 20 hours after transfection.

[0647] Preferably, the endonuclease in the form of mRNA is synthesized with a cap to enhance its stability according to techniques well known in the art, as described for example in Kore A.L. 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).

[0648] Typically, the electroporation step for transfecting pluripotent cells is typically performed in a closed chamber comprising parallel plate electrodes that generate a pulsed electric field between the parallel plate electrodes of greater than 100 volts / cm and less than 5,000 volts / cm that is substantially uniform throughout the treatment volume, such as described in WO / 2004 / 083379, which is incorporated by reference. Preferably, one such electroporation chamber has a geometric factor (cm 3 ) defined by the quotient of the square of the electrode gap (cm2) divided by the chamber volume (cm3) where the geometric factor is less than or equal to 0.1 cm 1 . 1wherein the suspension of cells and sequence-specific reagent is in a medium, which is adjusted such that the medium has an electrical conductivity in the range of 0.01 to 1.0 millisiemens. Typically, the cell suspension is subjected to one or more pulsed electric fields. By this method, the processing volume of the suspension is scalable and the processing time of the cells in the chamber is essentially uniform.

[0649] Due to their higher specificity, TALE-nucleases have proven to be particularly suitable sequence-specific nuclease reagents for therapeutic applications, especially in the form of heterodimers, i.e. working in pairs with a "right" monomer (also called "5" or "forward") and a "left" monomer (also called "3" or "reverse"), as reported, for example, by Mussolino et al. (TALE Nucleases for Targeted Gene Knockout and ® facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773).

[0650] As previously mentioned, the sequence-specific reagent is preferably in the form of a nucleic acid, such as DNA or RNA, encoding a rare-cutting endonuclease or a subunit thereof, but they can also be part of a conjugate involving a polynucleotide and a polypeptide, such as a so-called "ribonucleoprotein". Such conjugates can form with reagents such as Cas9 or Cpfl (RNA-guided endonucleases) or Argonaute (DNA-guided endonucleases), as recently described by Zetsche, B. et al. (Cpfl Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System (2015) Cell 163(3): 759-771) and by Gao F. et al. (DNA-guided genome editing using the Natronobacterium gregoryi Argonaute (2016) Nature Biotech), respectively, which involve RNA or DNA guides that can complex with their respective nuclease.

[0651] AAV vectors

[0652] Improving the efficiency of gene targeting insertion in DA neuronal cells using AAV vectors

[0653] The donor template is typically a polynucleotide sequence, which can be incorporated into various vectors described in the art to deliver the donor template into the nucleus where the endonuclease reagent becomes active to obtain its site-directed insertion into the genome, typically by NHEJ or homologous recombination.

[0654] Disclosed is a method of inserting an exogenous nucleic acid sequence into an endogenous polynucleotide sequence in a cell, the method comprising at least the steps of: transducing the cell with an AAV vector comprising the exogenous nucleic acid sequence and a sequence homologous to the targeted endogenous DNA sequence, and

[0655] inducing expression of a sequence-specific endonuclease reagent to cleave the endogenous sequence at the insertion locus.

[0656] The resulting insertion of the exogenous nucleic acid sequence can result in the introduction of genetic material, correction or replacement of an endogenous sequence, more preferably "in-frame" with respect to the endogenous gene sequence at the locus.

[0657] In some embodiments, each cell is transduced with 10 5 to 10 7 , preferably 10 6 to 10 7 , more preferably about 5.10 6 viral genomes. In some embodiments, the cells can be treated with a proteasome inhibitor, such as bortezomib, to further aid in homologous recombination. In some embodiments, the AAV vector used in the method can comprise a promoterless exogenous coding sequence so as to be placed under the control of an endogenous promoter at a locus selected from one of the loci listed in this specification. In some embodiments, the AAV vector used in the method can comprise a 2A peptide cleavage site followed by a cDNA (minus start codon), forming an exogenous coding sequence. Any AAV vector designed to carry out the methods described herein is disclosed, especially vectors comprising a sequence homologous to the insertion locus. Many other vectors known in the art, such as plasmids, episomal vectors, linear DNA substrates, etc., can also be used in accordance with the teachings herein.

[0658] As previously stated, the DNA vector comprises: (1) a foreign nucleic acid containing a foreign coding sequence to be inserted via homologous recombination, and (2) a sequence encoding a sequence-specific endonuclease reagent that facilitates the insertion. In some embodiments, the foreign nucleic acid under (1) does not contain any promoter sequence, while the sequence under (2) has its own promoter. In some embodiments, the nucleic acid under (1) contains an internal ribosome entry site (IRES) or a “self-cleaving” 2A peptide, such as T2A, P2A, E2A, or F2A, such that the endogenous gene into which the foreign coding sequence is inserted becomes polycistronic. The IRES of the 2A peptide may be before or after the foreign coding sequence.

[0659] transplant

[0660] In some implementations, GDNF-engineered DA neurons are transplanted into patients with rapidly progressing and mild Parkinson's disease. In some implementations, GBA / SNCA-engineered DA neurons are transplanted into patients to improve cognitive symptoms of Parkinson's disease.

[0661] In some implementations, engineered cells may be administered as a monotherapy to subjects in need. Engineered DA neurons, recombinant gene vectors, or gene-editing systems can be administered in a variety of ways. In some implementations, administration includes systemic, parenteral, intravenous, intracerebral, intraspinal, intrathecal, intracranial, intracisional, intraputral, intrahippocampal, intrastriatal, or intraventricular administration. In some implementations, administration includes intravenous, intracerebral, intraspinal, intrathecal, intracranial, intracisional, intraputral, intrastriatal, intrahippocampal, intrastriatal, or intraventricular injection. In some implementations, administration includes intrathecal injection with Threndlenburg tilt. In some implementations, administration includes direct injection into the substantia nigra pars compacta of the brain. In some implementations, administration includes introducing engineered DA neurons, recombinant gene vectors, or gene-editing systems into the brain or cerebrospinal fluid (CSF) of a subject.

[0662] The application of cells or cell populations can consist of the following: application of 10 4 -10 9 Cells / kg body weight, preferably 10 5 Up to 10 6 Cells / kg body weight, including all integer values ​​within those ranges. Administration of cells or cell populations may consist of doses of more than 10, typically more than 50, more usually more than 100, and typically more than 1000, comprising 10 cells derived from a single donor or patient sample. 6 Up to 10 8 Gene-edited cells.

[0663] In some embodiments, 1 x 10 9 -1 x 10 14 recombinant gene vector genomes per kilogram of subject body weight (vg / kg). In some embodiments, 1 x 10 9 -1 x 10 14 recombinant gene vector genomes per kilogram of subject body weight (vg / kg). In some embodiments, 1 x 10 9 -1 x 10 14 recombinant gene vector genomes per kilogram of subject body weight (vg / kg). In some embodiments, 1 x 10 7 -1 x 10 9 recombinant gene vector genomes per kilogram of subject body weight (vg / kg).

[0664] In some embodiments, a subject in need thereof receives treatment comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising engineered cells. In some embodiments, a subject in need thereof receives treatment comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising engineered cells comprising an exogenous GDNF, GBA, or SNCA gene or functional fragment thereof. The pharmaceutical composition can be administered intravenously. The pharmaceutical composition can be administered locally. In some embodiments, the method can further comprise administering one or more additional therapies. The one or more additional therapies can comprise transplantation. The one or more additional therapies can comprise immunotherapy. In some embodiments, the engineered cells can be autologous to the subject. In some embodiments, the engineered cells can be allogeneic to the subject.

[0665] The engineered DA neuronal cells can be administered to an individual in any amount or number (e.g., effective amount) that produces a detectable therapeutic or prophylactic benefit to the individual. In some embodiments, the dosage of engineered DA neuronal cells to be administered is simply the absolute number of cells, e.g., about 1 x 10 5 cells, 5 x 10 5 cells, 1 x 10 6 cells, 7 x 10 6 cells, 1 x 10 7 cells, 6 x 10 7 cells, 2 x 10 8 cells, 5 x 10 8 cells, 1 x 10 9 cells, 6 x 10 9 cells, 2 x 1010 about 1 x 105cells, 5 x 105cells, 1 x 106cells, 7 x 106cells, 1 x 107cells, 6 x 107cells, 2 x 108cells, 5 x 108cells, 1 x 109cells, 6 x 109cells, or 2 x 1010cells per kilogram of body weight of the subject to be treated. 10 11

[0666] In some embodiments, the engineered DA neuronal cells are administered to the subject in a cell number relative to the body weight of the subject to be treated, e.g., about 1 x 105cells, 5 x 105cells, 1 x 106cells, 7 x 106cells, 1 x 107cells, 6 x 107cells, 2 x 108cells, 5 x 108cells, 1 x 109cells, 6 x 109cells, or 2 x 1010cells per kilogram of body weight of the subject to be treated. 5 5 6 6 7 7 8 8 9 9

[0667] The cells or cell populations can be administered in one or more doses. In another embodiment, an effective amount of cells is administered as 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 judgment of the managing physician and depends on the clinical condition of the patient. The cells or cell populations can be obtained from any source, e.g., a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for particular diseases or conditions is within the skill of the art. An effective amount means an amount which provides the therapeutic or prophylactic benefit. The dose administered will depend on the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0668] In another embodiment, an effective amount of cells or compositions comprising those cells are administered parenterally. The administration can be intravenous administration.

[0669] The present disclosure also encompasses ways for detecting engineered cells comprising a desired genetic insertion, in particular performing a step of using a PCR method to detect the insertion of an exogenous coding sequence at an endogenous locus. In some embodiments, allogeneic tags can be used to monitor implant integrity and functionality.

[0670] The above written description provides a manner and process of making and using it, so that any person skilled in the art can prepare and use it, and the support provided by this disclosure is particularly directed to the subject matter of the appended paragraphs which form a part of the original description.

[0671] Combination therapy

[0672] ​​​​​​​​​​​​The modified DA neuronal cell compositions can also be used in combination with other therapeutically valuable agents for the treatment of PD symptoms. According to embodiments, the treatment can be administered to patients undergoing immunosuppressive therapy. Generally, the other agents do not necessarily have to be administered in the same pharmaceutical composition, and due to different physical and chemical properties, can preferably be administered by different routes. Where possible, the determination of the mode of administration and the desirability of administration in the same pharmaceutical composition is well within the knowledge of the skilled clinician. Initial administration can be made according to established protocols known in the art, and thereafter, based on observed effects, the skilled clinician can modify the dosage, mode of administration, and timing of administration.

[0673] In some embodiments, the treatment with the modified DA neuronal cell compositions can be used in conjunction with drugs designed to achieve cholinergic systems in the brain. Examples of such drugs can be, but are not limited to, the group comprising or consisting of: donepezil (Aricept), rivastigmine (Exelon), and galantamine (Razadyne).

[0674] It is known to those skilled in the art that the therapeutically effective dose can vary when drugs are used in therapeutic combinations. Methods for experimentally determining the therapeutically effective dose of drugs and other agents for combination therapy regimens are described in the literature. For example, metronomic dosing (i.e., providing more frequent, lower doses in order to minimize toxic side effects) has been extensively described in the literature. Combination therapy further includes periodic treatment that is initiated and stopped at different times to assist in the clinical management of the patient.

[0675] For combination therapy, the dosage of the co-administered therapeutic agents will, of course, vary depending on the type of co-agent employed, on the particular modified DA neuronal cell, and on the Parkinson's disease and other secondary parkinsonian conditions to be treated.

[0676] It will be appreciated that dosage regimens for treating, preventing, or ameliorating the conditions for which relief is sought can be modified, depending on a variety of factors. These factors include the conditions from which the subject suffers, as well as the age, body weight, sex, diet, and general medical condition of the subject. Accordingly, the dosage regimen actually employed can vary widely, and therefore can deviate from the dosage regimens set forth herein.

[0677] The modified DA neuronal cells and additional therapeutic agents that make up the combination therapy disclosed herein can be in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The agents that make up the combination therapy can also be administered sequentially, where either therapeutic compound is administered by a regimen requiring two steps of administration. The two-step administration regimen can require sequential administration of the active agents or separate administration of the individual active agents. The time period between the multiple steps of administration can range from a few minutes to several hours depending on the properties of each agent, such as potency, solubility, bioavailability, plasma half-life, and kinetic profile of the agent. Diurnal variations in various physiological parameters can also be assessed to determine the optimal dosage interval.

[0678] The initial administration can be via any feasible route, such as, for example, intravenous injection, bolus, infusion over 5 minutes to about 5 hours, pill, capsule, inhaler, injection, transdermal patch, buccal delivery, etc., or combinations thereof. The compounds should be administered as soon as possible after the onset of the disease or condition is detected or suspected, and for a length of time necessary to treat or prevent the PD condition.

[0679] Dosage forms

[0680] Useful compositions can be formulated for administration to a subject via any of the usual modalities, such as, for example, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), buccal, inhalation, intranasal, rectal, or transdermal routes of administration.

[0681] Pharmaceutical compositions comprising the modified DA neuronal cells, alone or in combination with one or more other therapeutic agents, can be formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, mists, gels, syrups, elixirs, slurries, suspensions, etc., for oral ingestion by the patient to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, lozenges, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0682] Pharmaceutical formulations for oral use can be obtained by mixing one or more solid excipient with one or more compounds, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if required, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, microcrystalline cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and / or other excipients such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents can be added, such as a cross-linked sodium carboxymethylcellulose, a poly- vinylpyrrolidone, agar, or a salicylic acid, its salts or sodium carbonate.

[0683] In another aspect, the dosage form can include a microencapsulated formulation. In some embodiments, one or more other compatible materials are present in the microencapsulated material. Exemplary materials include, but are not limited to, pH adjusting agents, erosion promoters, antifoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0684] Microencapsulated formulations of DA neuronal cell populations can be formulated by methods known to those of ordinary skill in the art. Such known methods include, for example, spray-drying, spinning disk-solvent, hot-melt, spray-chilling, fluidized bed, electrostatic deposition, centrifugal extrusion, spinning suspension separation, liquid-gas or solid-gas interfacial polymerization, pressure extrusion, or spray solvent extraction bath. In addition to these, several chemical techniques can also be used, for example, complex coacervation, solvent evaporation, polymer-polymer incompatibility, interfacial polymerization in a liquid medium, in situ polymerization, intraliquid drying, and desolvation in a liquid medium. In addition, other methods such as roller compaction, extrusion / spheronization, coacervation, or nanoparticle coating can also be used.

[0685] 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 from the dosage form into which they are incorporated according to a desired profile over an extended period of time. Controlled release profiles include, for example, sustained release, extended release, pulsatile release, and delayed release profiles. In contrast to immediate release compositions, controlled release compositions allow for the delivery of a pharmaceutical agent to a subject according to a predetermined profile over an extended period of time. Such release rates can provide therapeutically effective levels of a pharmaceutical agent over an extended period of time, thereby providing a pharmacological response for a longer period of time with minimized side effects as compared to conventional fast release dosage forms. Such longer periods of response provide inherent benefits that are not achievable with corresponding short-acting immediate release formulations.

[0686] In some embodiments, the solid dosage form can be formulated as an enteric-coated delayed release oral dosage form, i.e., as an oral dosage form of a pharmaceutical composition that utilizes an enteric coating to affect release in the small intestine of the gastrointestinal tract. The enteric-coated dosage form can be a compressed or molded or extruded tablet / mold (coated or uncoated) containing granules, powders, pellets, beads, or microparticles of the active ingredient and / or other composition components, which are themselves coated or uncoated. The enteric-coated oral dosage form can also be a capsule (coated or uncoated) containing a solid carrier or pellets, beads, or granules of the composition, which are themselves coated or uncoated.

[0687] As used herein, the term "delayed release" refers to delivery such that release can be accomplished at some generally predictable location in the gastrointestinal tract that is more distal than would have been accomplished in the absence of the delayed release modification. In some embodiments, the method for delayed release is a coating. Any coating should be applied to a sufficient thickness such that the entire coating does not dissolve in a gastrointestinal fluid having a pH less than about 5, but does dissolve at a pH of about 5 and above. Any anionic polymer that exhibits a pH dependent dissolution profile is contemplated to be useful as an enteric coating in the methods and compositions to achieve delivery to the lower gastrointestinal tract. In some embodiments, the polymer is an anionic carboxylic acid polymer.

[0688] In some embodiments, the coating can and typically does contain a plasticizer and possibly other coating excipients such as colorants, talc, and / or magnesium stearate, which are well known in the art. Suitable plasticizers include Citroflex 2, triacetin (glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerin, fatty acid esters, propylene glycol, and dibutyl phthalate. In particular, the anionic carboxylic acid acrylate polymers can typically contain 10-25% by weight of a plasticizer, especially dibutyl phthalate, polyethylene glycol, triethyl citrate, and triacetin. The coating is applied using conventional coating techniques such as spray or pan coating. The coating thickness must be sufficient to ensure that the oral dosage form remains intact until reaching the desired site of localized delivery in the intestinal tract.

[0689] Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

[0690] Aqueous suspensions and dispersions can remain homogeneous for at least 4 hours as defined in The USP Pharmacists' Pharmacopeia (2005 Edition, Chapter 905). Homogeneity should be determined by a sampling method consistent with determining homogeneity of the entire composition. In one embodiment, an aqueous suspension can be resuspended into a homogeneous suspension by physical agitation for less than 1 minute. In another embodiment, an aqueous suspension can be resuspended into a homogeneous suspension by physical agitation for less than 45 seconds. In yet another embodiment, an aqueous suspension can be resuspended into a homogeneous suspension by physical agitation for less than 30 seconds. In another embodiment, no agitation is required to maintain a homogeneous aqueous dispersion.

[0691] In addition to the additives listed above, the liquid formulations can also include inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers. Exemplary emulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3- butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodium doccusate, cholesterol, cholesterol esters, taurolidine, phosphotidylcholine, oils including cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, mixtures of these, and the like.

[0692] Injectable formulations

[0693] Formulations suitable for intramuscular, subcutaneous, or intravenous injection can include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Formulations suitable for subcutaneous injection can also contain additives such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absoφtion of injectable pharmaceutical forms can be brought about by the use of agents delaying absoφtion, such as aluminum monostearate and gelatin.

[0694] For intravenous injection, the compounds can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, suitable formulations can include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally known in the art.

[0695] Parenteral injections can involve bolus or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0696] The pharmaceutical compositions can be in unit dosage form suitable for single administration of precise dosages. In unit dosage form, the preparation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in packaged form, e.g., a tablet or capsule, and a powder in a vial or ampoule. An aqueous suspension composition can be packaged in a single-dose non-reclosable container. Alternatively, a multiple-dose reclosable container can be used, in which case it is common to include a preservative in the composition. By way of example only, formulations for parenteral injection can be in unit dosage form, including, but not limited to, an ampoule, or in a multi-dose container with a preservative added to the formulation.

[0697] Therapeutic outcomes

[0698] In some embodiments, the number of neuronal mature cells in the subject after the administering step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is greater than the number of neuronal mature cells in the subject before the administering step. In some embodiments, the number of endogenous neuronal mature cells in the subject after the administering step is greater than the number of endogenous neuronal mature cells in the subject before the administering step.

[0699] In some embodiments, the number of dopaminergic neurons in the subject after the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is greater than the number of dopaminergic neurons in the subject before the administration step. In some embodiments, the level of dopamine in the subject after the administration step is greater than the level of dopamine in the subject before the administration step. In some embodiments, 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 so treated. In some embodiments, the level of dopamine in a subject treated by the methods disclosed herein (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is increased compared to the level of dopamine in a subject not so treated. In some embodiments, the level of dopamine in the substantia nigra of a subject treated by the methods disclosed herein is increased compared to the level of dopamine in the substantia nigra of a subject not so treated. In some embodiments, the level of GDNF in the cerebrospinal fluid (CSF) of the subject after the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is greater than the level of GDNF in the CSF of the subject before the administration step. In some embodiments, the level of GBA in the CSF of the subject after the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is greater than the level of GBA in the CSF of the subject before the administration step. In some embodiments, the ratio of GBA to SNCA in the subject after the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is less 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 the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is greater than the ratio of GBA to SNCA in the subject before the administration step.

[0700] In some embodiments, the ratio of GBA protein to alpha-synuclein in the subject after the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is less 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 in the subject after the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is greater than the ratio of GBA protein to alpha-synuclein in the subject before the administration step.

[0701] In some embodiments, the subject’s Unified Parkinson’s Disease Rating Scale (UPDRS) score prior to the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) is improved compared to the subject’s UPDRS score prior to the administration step. In some embodiments, after the administration step, the subject’s neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies.

[0702] In some embodiments, the subject’s cognitive symptoms prior to the administration step (administering the engineered DA neuronal cells, the recombinant gene vector, or the gene editing system) are improved compared to the subject’s cognitive symptoms prior to the administration step.

[0703] In some embodiments, the long-term engraftment integrity of the engineered cells administered to the subject is improved compared to the long-term engraftment integrity of non-engineered cells administered to the subject.

[0704] Sequence

[0705] In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF gene (or vector) comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-10, respectively.

[0706] In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11-20, respectively.

[0707] In some embodiments, the gene is a GDNF gene, and the wild-type GDNF protein comprises the amino acid sequence set forth in any one of SEQ ID NO: 20.

[0708] In some embodiments, the gene is a GBA gene, and the wild-type GBA protein comprises the amino acid sequence set forth in any one of SEQ ID NO: 17.

[0709] In some embodiments, the polynucleotide comprises a sequence that is at least 70%, 75%, 80%, 85%, 95%, or 99% identical to a PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, or GDNF polynucleotide sequence set forth in SEQ ID NOs: 1-10, 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 between 5 and 20 CpG islands.

[0710] The polynucleotide sequences of the vectors, donor templates comprising exogenous coding sequences and / or sequences homologous to an endogenous locus, sequences related to the resulting engineered cells, and sequences that allow for detection of the engineered cells are all part of the present disclosure.

[0711] In some embodiments, the transgene encodes a GDNF gene, and the transgene polynucleotide sequence shares at least 95% identity to one of SEQ ID NOs: 10, and is positioned after a promoter selected from SEQ ID NOs: 21-30.

[0712] In some embodiments, the transgene encodes a GDNF gene, and the transgene polynucleotide sequence shares at least 95% identity to one of SEQ ID NOs: 10, and is positioned after a promoter selected from SEQ ID NOs: 21-30.

[0713] In some embodiments, the transgene encodes a SNCA gene, and the transgene polynucleotide sequence shares at least 95% identity to one of SEQ ID NOs: 8, and is positioned after a promoter selected from SEQ ID NOs: 21-40 or 43.

[0714] In some embodiments, the first transgene encodes GBA, and the second transgene encodes SNCA, and the first transgene is positioned after a promoter selected from SEQ ID NOs: 31-40 or 43, and the second transgene is positioned after a promoter selected from SEQ ID NOs: 21-40 or 43.

[0715] Genetic sequences encoding GDNF, GBA, and / or SNCA

[0716] The GDNF, GBA and / or SNCA sequences can be introduced at selected genetic loci by targeted gene recombination, more specifically under the control of an endogenous promoter. In some embodiments, preferably, exogenous GDNF is expressed only after DA neuronal cells have differentiated and implant function has been established, and preferably, exogenous GBA and SNCA + / - are expressed immediately after the engineered cells are transplanted into the patient.

[0717] According to embodiments, the exogenous sequences encode a polypeptide displaying 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 the endogenous coding sequence present at the locus (knocking out by knock-in) such that gene inactivation can be combined with gene occurrence.

[0718] In some embodiments, the exogenous sequences can be introduced into the genome without deleting or modifying the endogenous coding sequence present at the locus. This occurs whether the endogenous coding sequence is in vitro pluripotent cells or in vivo.

[0719] Depending on the targeted locus and its involvement in DA neuronal cell activity, the targeted endogenous gene can be unexpressed or maintain its original function, whether the targeted gene is in vitro cells or in vivo. If the targeted gene is essential for DA neuronal cell activity or differentiation, the insertion procedure can generate a single (Kl) without gene inactivation. Alternatively, if the targeted gene is considered to be involved in DA neuronal cell differentiation, the insertion procedure is designed to prevent the expression of the endogenous gene, preferably by knocking out the endogenous sequence, while enabling the expression of the introduced exogenous coding sequence.

[0720] In some embodiments, the method relies on upregulation of target gene expression in various kinetic ways after activation of the GDNF / GBA signaling pathway and / or modulation of the SNCA signaling pathway by targeted integration (with or without natural gene disruption) at specific loci (e.g., as non-limiting examples, SEQ ID NOs: 21-40 or 43).

[0721] In some embodiments, engineered DA neuronal cells, and preferably DA neuronal cells for infusion into a patient, are disclosed that comprise an exogenous sequence encoding a PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA+ / - or GDNF polypeptide that is integrated at the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA+ / - or GDNF endogenous loci for expression of them under the control of the endogenous promoters present at these loci. In some embodiments, the endogenous promoters comprise or consist of SEQ ID NOs: 21-40 or 43.

[0722] The engineered DA neuronal cells can be SNCA + / + , SCNA - / - or SCNA + / - depending on the therapeutic indication and the recipient patient. In some embodiments, the engineered DA neuronal cells are further rendered T cell receptor negative for allogeneic transplantation.

[0723] The gene editing step of integrating an exogenous sequence encoding a 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 engineered DA neuronal cells.

[0724] According to embodiments, the method relies on the introduction of the sequence-specific endonuclease reagent and / or the donor template containing the gene of interest and the sequence homologous to the target gene by transfection of ssDNA (oligonucleotide as non-limiting example), dsDNA (plasmid DNA as non-limiting example) and more particularly adeno-associated virus (AAV) as non-limiting example.

[0725] Kit

[0726] Disclosed are kits for pluripotent and / or DA neuronal cell transfection comprising a polynucleotide encoding a sequence-specific endonuclease reagent and a donor sequence designed to integrate an exogenous sequence at the locus targeted by the reagent. Examples of such kits are kits comprising an mRNA encoding a rare-cutting endonuclease targeting synapsin 1, DAT, VMAT, TH, AADC, a tamoxifen-inducible promoter, a RU486-inducible promoter, or other promoters that allow time or small molecule control of the locus and an AAV vector containing an exogenous sequence encoding GDNF; kits comprising an mRNA encoding a rare-cutting endonuclease targeting synapsin 1, DAT, VMAT, TH, AADC, a tamoxifen-inducible promoter, a RU486-inducible promoter, or other promoters that allow time control or small molecule control of the locus and an AAV vector containing an exogenous sequence encoding GBA.

[0727] Disclosed are kits for pluripotent and / or DA neuronal cell transfection comprising a polynucleotide encoding a sequence-specific endonuclease reagent and an exogenous polynucleotide sequence, in some embodiments, incorporated into an AAV vector is an exogenous sequence comprising a sequence encoding GBA, GBA and / or SCNA + / - or a functional fragment or variant thereof.

[0728] EMBODIMENT

[0729] EMBODIMENT 1

[0730] Applicants identified genomic safe harbor (GSH) sites suitable for iPSC gene editing and expression in dopaminergic neurons to enable development of a novel secreted GNDF stem cell replacement therapy for Parkinson’s disease. See Figure 2 for identification of GSH sites based on computational research and additional predictive and validation modeling. Based on these results, GDNF can be inserted into a GSH in iPSCs and novel cell and gene therapies for treating Parkinson’s disease can be identified.

[0731] scATAC-seq data was generated from clinically compliant iPSC lines and resulting dopaminergic neuronal progenitor drug product RNDP-001. This data was compared to published ATAC-seq data from dopaminergic neurons isolated from human healthy brains and Parkinson’s brains to define shared regions of chromatin accessibility based on catalog data of GSH site suitability. First, Applicants defined 1,875 novel GSH candidates that were identified based on being distal from genes, regulatory regions, telomeres, and centromeres to prevent transgene interference with gene expression, division, and function. The main challenge for precisely defining where to target the transgene was that these 1,875 putative GSH regions span > 142 million nucleotides of genomic space, necessitating the implementation of additional prioritization criteria.

[0732] The top predicted sgRNAs can be selected for editing using published tools to predict sgRNAs with the highest on-target activity and lowest off-target activity and empirically defining the best cutting sgRNAs. The lead sgRNA can then be inserted into hGDNF using a DNA donor template into the identified genomic safe harbor (GSH). Single cell clones can also be isolated and tested for safety and GSH accessibility.

[0733] The selected clones can then be differentiated into dopaminergic progenitor cells and post-mitotic neurons to measure GDNF secretion. Existing release criteria involving a panel of flow cytometry and ddPCR assays are used to establish whether the edited iPSCs have the ability to have the correct dopaminergic progenitor cell fate specification. RNAseq profiling can also be performed to assess the impact of GDNF insertion on differentiation capacity.

[0734] Genomic accessibility can be used to narrow down the best GSH loci for GDNF insertion into iPSCs with the ultimate goal of expression in post-mitotic dopaminergic neurons.

[0735] Figure 3 The starting point for GSH selection is shown, which can be further selected based on publicly available criteria including sub-region selectivity.

[0736] The present disclosure can be better understood by the following numbered paragraphs:

[0737] 1. An engineered cell comprising at least one sequence encoding a transgene, wherein the at least one sequence is inserted within a safe harbor locus, the safe harbor locus being located at any one or more of the loci provided in Table 1.

[0738] 2. An engineered cell comprising at least one sequence encoding a transgene, wherein the at least one sequence is inserted into a safe harbor locus, which safe harbor locus is located at any one or more loci provided in Table 1; and wherein expression of the at least one sequence encoding a transgene is operably linked to an endogenous promoter or an exogenous promoter.

[0739] 3. An engineered cell comprising at least one sequence encoding a transgene, wherein the at least one sequence is inserted into a safe harbor locus, which safe harbor locus is located at any one target locus in Table 1; and wherein expression of the at least one sequence encoding a transgene is operably linked to an endogenous promoter or an exogenous promoter, and wherein the engineered cell is undifferentiated.

[0740] 4. The engineered cell of paragraph 1 or 2 or 3, wherein the target locus is selected from the group consisting of: 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; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184111383-184112593; chr3: 184chr2: 147082542-147083491; chr2: 180125287-180126295; chr2: 121974721-121975718; chr2: 122660609-122661835; chr2: 75920596-75921844; chr2: 103401408-103402401; chr2: 133843863-133844997; chr2: 180123973-180124967; chr2: 160582472-160583418; chr20: 55667282-55669174; chr22: 49103355-49104388; chr3: 67113422-67114525; chr3: 74614218-74615242; chr3: 5426026-5426982; chr3: 117399281-117400399; chr3: 28915343-28917419; chr3: 117439560-117440548; chr3: 117237013-117239704; chr3: 137162914-137164165; chr3: 106609403-106610465; chr3: 67109328-67110455; chr3: 16048355-16049420; chr3: 117397641-117399034; chr3: 104908816-104909868; chr3: 104596179-104597355; chr4: 180058397-180059507; chr4: 180057145-180058232; chr4: 27684257-27685441; chr4: 116925637-116926672; chr4: 166165437-166166501; chr4: 156168620-156169693; chr4: 18689105-18690087; chr4: 85370123-85371213; chr4: 64537927-64540013; chr4: 154982074-154983202; chr4: 130563237-130564339; chr5: 71937993-71940439; chr5: 113806007-113808129;chr5: 18393819-18394795; chr5: 18465687-18466781; chr5: 123874455-123875520; chr5: 123875580-123876580; chr5: 71932489-71933717; chr5: 34474174-34475918; chr5: 18113286-18115121; chr5: 103808080-103809225; chr5: 144598204-144599350; chr5: 108521605-108522526; chr5: 113692453-113694380; chr5: 101678253-101679223; chr5: 87944237-87945265; chr5: 101389859-101390987; chr5: 113678518-113679693; chr5: 88089287-88091255; chr5: 121797421-121798482; chr5: 87949701-87952291; chr5: 103894512-103895553; chr5: 63401153-63402160; chr6: 85922657-85923735; chr6: 104472033-104473578; chr6: 104525429-104526360; chr6: 47097062-47097998; chr6: 87968250-87969278; chr6: 16964556-16965560; chr6: 137415153-137416177; chr6: 91081186-91082896; chr6: 99746005-99747236; chr6: 99822427-99823526; chr6: 140439792-140441984; chr6: 137313918-137314874; chr6: 48765024-48766063; chr6: 90890535-90891762; chr7: 152925055-152926297; chr7: 12111174-12112347; chr7: 42493270-42494306; chr7: 31192574-31193687; chr7: 22000950-22002150;chr7: 96785947-96787328; chr8: 141715497-141716580; chr8: 26189940-26191013; chr8: 131803831-131805003; chr8: 106856111-106857217; chr8: 75975412-75977242; chr8: 115076228-115077322; chr8: 131815871-131817817; chr8: 137492454-137493716; chr9: 85258346-85259505; chr9: 17904998-17907161; chr9: 78439098-78440151; chr9: 16132791-16134459; chr9: 29513212-29515120; chr9: 75265233-75266237; chr9: 7542343-7543943; chr9: 118169738-118170714; chr9: 71517517-71519983; chr9: 7401713-7402906; chr9: 26325995-26327156; chr9: 1453442-1454897; chr9: 105156511-105157922; chr9: 12425914-12426947; chrX: 68894085-68895495; chrX: 20530099-20531285; chrX: 40996550-40997770; chrX: 20527023-20528155; chrX: 94058277-94059471; or chrX: 138127607-138128791.

[0741] 5. The engineered cell of paragraphs 1-4, wherein the endogenous promoter is any one of SEQ ID NOs: 21-40 or 43.

[0742] 6. The engineered cell of any one of paragraphs 2-5, wherein the endogenous promoter is expressed in a mature neuronal cell type.

[0743] 7. The engineered cell of any one of paragraphs 1-6, wherein the engineered cell is a stem cell, a pluripotent cell, an iPSC, a human cell, a primary cell, a DA neuronal cell, a DA neuronal cell progenitor, or a combination thereof.

[0744] 8. The engineered cell of any one of paragraphs 1-7, wherein the engineered cell is undifferentiated.

[0745] 9. The engineered cell of any one of paragraphs 1-8, wherein the engineered cell is capable of differentiating into a DA neuronal cell.

[0746] 10. The engineered cell of any one of paragraphs 1-9, wherein the transgene encodes a gene selected from the group consisting of, consisting of, or consisting essentially of GDNF, GBA PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof.

[0747] 11. A composition comprising the engineered cell of any one of paragraphs 1-10 and a pharmaceutical excipient.

[0748] 12. A guide ribonucleic acid (gRNA) for editing a cell at a safe harbor locus located at a locus in Table 1.

[0749] 13. A method of editing a cell having chromosomal DNA, the method comprising inserting at least one sequence encoding a transgene within a safe harbor locus in the chromosomal DNA of the cell, wherein the safe harbor locus is any one or more of the target loci provided in Table 1.

[0750] 14. A method of editing a pluripotent cell, the method comprising contacting a pluripotent cell 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 insertion of the at least one sequence into chromosomal DNA within a safe harbor locus, wherein the safe harbor locus is selected from any one of the target loci in Table 1.

[0751] 15. The method of paragraph 13 or 14, wherein the target locus is selected from the group consisting of: chr1: 214186905-214187961; chr1: 91164906-91165855; chr1: 88180241-88181223; chr1: 72546731-72548018; chr1: 199684394-199685410; chr1: 104647871-104648861; chr10: 109456554-109457498; chr10: 84613037-84614143; chr10: 128540929-128541943; chr10: 128693340-128695044; chr10: 36778899-36780002; chr10: 36725218-36726233; chr11: 121721801-121724194; chr11: 42524286-42525334; chr11: 81647375-81648903; chr11: 116192498-116194401; chr11: 114774331-114775325; chr11: 128021283-128022329; chr11: 116224105-116225916; chr11: 20309899-20311995; chr12: 92649604-92650584; chr12: 94763797-94764936; chr13: 103882813-103883879; chr13: 52967785-52968920; chr13: 52904228-52906942; chr13: 59317916-59319213; chr13: 70309932-70311460; chr13: 76473185-76474229; chr15: 96947520-96948565; chr16: 66127013-66128412; chr17: 56715590-56716621; chr18: 61088416-61089493; chr18: 40576326-40578671; chr18: 27749740-27750778; chr18: 67199846-67201011; chr2: 133886374-133887591; chr2: 163347709-163348850;chr2: 147082542-147083491; chr2: 180125287-180126295; chr2: 121974721-121975718; chr2: 122660609-122661835; chr2: 75920596-75921844; chr2:103401408-103402401; chr2: 133843863-133844997; chr2: 180123973-180124967;chr2: 160582472-160583418; chr20: 55667282-55669174; chr22: 49103355-49104388; chr3: 67113422-67114525; chr3: 74614218-74615242; chr3: 5426026-5426982; chr3: 117399281-117400399; chr3: 28915343-28917419; chr3: 117439560-117440548; chr3: 117237013-117239704; chr3: 137162914-137164165; chr3:106609403-106610465; chr3: 67109328-67110455; chr3: 16048355-16049420; chr3:117397641-117399034; chr3: 104908816-104909868; chr3: 104596179-104597355;chr4: 180058397-180059507; chr4: 180057145-180058232; chr4: 27684257-27685441; chr4: 116925637-116926672; chr4: 166165437-166166501; chr4:156168620-156169693; chr4: 18689105-18690087; chr4: 85370123-85371213; chr4:64537927-64540013; chr4: 154982074-154983202; chr4: 130563237-130564339;chr5: 71937993-71940439; chr5: 113806007-113808129;chr5: 18393819-18394795; chr5: 18465687-18466781; chr5: 123874455-123875520; chr5: 123875580-123876580; chr5: 71932489-71933717; chr5: 34474174-34475918; chr5: 18113286-18115121; chr5: 103808080-103809225; chr5: 144598204-144599350; chr5: 108521605-108522526; chr5: 113692453-113694380; chr5: 101678253-101679223; chr5: 87944237-87945265; chr5: 101389859-101390987; chr5: 113678518-113679693; chr5: 88089287-88091255; chr5: 121797421-121798482; chr5: 87949701-87952291; chr5: 103894512-103895553; chr5: 63401153-63402160; chr6: 85922657-85923735; chr6: 104472033-104473578; chr6: 104525429-104526360; chr6: 47097062-47097998; chr6: 87968250-87969278; chr6: 16964556-16965560; chr6: 137415153-137416177; chr6: 91081186-91082896; chr6: 99746005-99747236; chr6: 99822427-99823526; chr6: 140439792-140441984; chr6: 137313918-137314874; chr6: 48765024-48766063; chr6: 90890535-90891762; chr7: 152925055-152926297; chr7: 12111174-12112347; chr7: 42493270-42494306; chr7: 31192574-31193687; chr7: 22000950-22002150;chr7: 96785947-96787328; chr8: 141715497-141716580; chr8: 26189940-26191013; chr8: 131803831-131805003; chr8: 106856111-106857217; chr8: 75975412-75977242; chr8: 115076228-115077322; chr8: 131815871-131817817; chr8: 137492454-137493716; chr9: 85258346-85259505; chr9: 17904998-17907161; chr9: 78439098-78440151; chr9: 16132791-16134459; chr9: 29513212-29515120; chr9: 75265233-75266237; chr9: 7542343-7543943; chr9: 118169738-118170714; chr9: 71517517-71519983; chr9: 7401713-7402906; chr9: 26325995-26327156; chr9: 1453442-1454897; chr9: 105156511-105157922; chr9: 12425914-12426947; chrX: 68894085-68895495; chrX: 20530099-20531285; chrX: 40996550-40997770; chrX: 20527023-20528155; chrX: 94058277-94059471; or chrX: 138127607-138128791.

[0752] 16. The method of any one of paragraphs 13-15, wherein the at least one sequence comprises an exogenous promoter of any one of Seq ID no. 21-40 or 43.

[0753] 17. The method of any one of paragraphs 13-16, wherein the cell is a stem cell, a pluripotent cell, an iPSC, a human cell, a primary cell, a DA neuronal cell, a DA neuronal progenitor cell, or a combination thereof.

[0754] 18. The method of any one of paragraphs 13-17, wherein the engineered cell is undifferentiated.

[0755] 19. The method of any one of paragraphs 13-18, wherein the at least one sequence is inserted using homology directed repair.

[0756] 20. The method of any one of paragraphs 13-19, wherein the at least one sequence is inserted using homology independent targeted insertion.

[0757] 21. The method of any one of paragraphs 13-20, wherein the at least one sequence is inserted using one or more guide ribonucleic acids (gRNAs) and one or more Cas9 endonucleases.

[0758] 22. The method of any one of paragraphs 13-21, wherein the transgene is selected from the group comprising GDNF, GBA PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof.

[0759] 23. An ex vivo method of obtaining an engineered cell or population thereof, comprising: a. obtaining a cell; b. genetically modifying the cell by inserting at least one sequence encoding a transgene within a safe harbor locus, wherein the safe harbor locus is selected from any one of the target loci in Table 1.

[0760] 24. The method of paragraph 23, wherein obtaining a cell comprises: (i) collecting a tissue sample from a subject, (ii) isolating the cell from the tissue sample, and (iii) culturing the cell in vitro.

[0761] 25. The method of paragraph 23, wherein the tissue sample is a blood sample.

[0762] 26. The method of any one of paragraphs 23-25, wherein the cell is a stem cell, a human cell, a primary cell, a hematopoietic cell, an adaptive immune cell, an innate immune cell, a T cell, or a T cell progenitor cell, or combinations thereof.

[0763] 27. The method of any one of paragraphs 23-26, wherein the engineered cell is undifferentiated.

[0764] 28. The method of any one of paragraphs 23-27, wherein the 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.

[0765] 29. The method of any one of paragraphs 23-28, wherein the at least one sequence is inserted using homology directed repair.

[0766] 30. The method of any one of paragraphs 23-29, wherein the at least one sequence is inserted using homology independent targeted insertion.

[0767] 31. The method of any of paragraphs 23-30, wherein the genetic modification in step (b) comprises contacting the cell with one or more guide ribonucleic acid (gRNA), the at least one sequence, and one or more Cas9 endonuclease, wherein the one or more gRNA and Cas9 endonuclease facilitate insertion of the at least one sequence into the chromosomal DNA within the safe harbor locus.

[0768] 32. The method of any of paragraphs 23-31, wherein the transgene encodes a gene selected from the group comprising GDNF, GBA PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof.

[0769] 33. The method of any of paragraphs 23-32, wherein the at least one sequence comprises an exogenous promoter, and the exogenous promoter is operably linked to the transgene.

[0770] 34. The method of any of paragraphs 23-33, wherein the exogenous promoter is any one of SEQ ID NOs: 21-40 or 43.

[0771] 35. A method of treating a subject having or at risk of having a disease, comprising administering to the subject an effective amount of the cell of any of paragraphs 13-24, population thereof, or the composition of paragraphs 1-12.

[0772] 36. A method of treating a subject having or at risk of having a disease, comprising: a. performing the method of any of paragraphs 13-35; and b. administering to the subject an effective amount of a composition comprising the cell or population thereof.

[0773] 37. The method of paragraph 35 or 36, wherein the composition is administered to the subject by infusion.

[0774] 38. The method of paragraphs 35-37, wherein the disease is Parkinson’s disease.

[0775] 39. The method of any of paragraphs 35-38, wherein the disease is a disorder associated with central nervous system degeneration.

[0776] 40. A method of treating a subject having or at risk of having a disease, comprising administering to the subject an effective amount of the cell of any of paragraphs 1-11, population thereof, or the composition of paragraph 12.

[0777] 41. A method of treating a subject having or at risk of having a disease, comprising: c. performing the method of any one of paragraphs 13-34; and d. administering to the subject an effective amount of a composition comprising the cell or population thereof.

[0778] 42. The method of paragraphs 40-41, wherein the composition is administered to the subject by infusion.

[0779] 43. The method of paragraphs 40-42, wherein the disease is Parkinson’s disease.

[0780] 44. A method of identifying a safe harbor locus, comprising: a. identifying a gene or non-coding region in a chromosome above a threshold level of chromatin accessibility; b. generating a model correlating the gene or non-coding region from step (a) with published ATAC-seq data from dopaminergic neurons isolated from human healthy brains and Parkinsonian brains with respect to the chromosome; and c. selecting a safe harbor locus based on a threshold parameter; wherein the safe harbor locus is selected for insertion of at least one sequence encoding a transgene within a cell.

[0781] 45. The method of paragraph 44, wherein the threshold parameter comprises one or more of: stable expression of a transgene, a gene knockout that confers a benefit to cell function, no known function within a cell, stable transgene expression in vitro, negligible off-target cleavage as detected by iGuide-Seq or CRISPR-Seq, fewer off-target cleavages relative to other loci as detected by iGuide-Seq or CRISPR-Seq, negligible transgene-independent cellular toxicity, negligible transgene-independent cytokine expression, negligible transgene-independent chimeric antigen receptor expression, negligible de-regulation or silencing of nearby genes, peak size data generated with processed ATAC-seq data, profile data generated with processed ATAC-seq data, highest efficiency of transgene insertion, persistent expression after extended culture, and lack of oncogenic gene expression.

[0782] 46. The method of any one of paragraphs 44-45, wherein chromatin accessibility is measured using transposase accessible chromatin sequencing (ATAC-seq) assay.

[0783] 47. The method of any one of paragraphs 44-46, wherein the engineered cell is a stem cell, a pluripotent cell, an iPSC, a human cell, a primary cell, a DA neuronal cell, a DA neuronal progenitor cell, or a combination thereof.

[0784] 48. The engineered cell, composition, or method of any of the preceding paragraphs, wherein the insertion within the safe harbor locus increases GDNF secreted protein production, GDNF secreted protein is produced and taken up by endogenous cells after the administered engineered cell has matured into a neuronal mature cell type.

[0785] 49. The engineered cell, composition, or method of any of the preceding paragraphs, wherein the knock-in efficiency at any of the safe harbor loci in Table 1 is increased relative to other locations along the chromosome.

Claims

1. An engineered cell population, wherein the engineered cells comprise: at least one exogenous GBA gene or a functional fragment thereof.

2. The cell population of claim 1, wherein the engineered cells are human DA neurons.

3. The cell population of claim 1, wherein the human DA neurons are derived from pluripotent cells.

4. The cell population of claim 1, wherein the engineered cells are ineffectively engineered to be SNCA hemizygotes.

5. The cell population of claim 1, wherein the exogenous GBA gene or a functional fragment thereof is located within or near a safe harbor locus.

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

7. The cell population of claim 5, wherein the safe harbor locus is selected from 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: 70309932-70311460; chr13: 76473185-76474229; chr15: 96947520-96948565; chr16: 66127013-66128412; chr17: 56715590-56716621;chr18: 61088416-61089493; chr18: 40576326-40578671; chr2: 133886374-133887591; chr2: 163347709-163348850;chr2: 147082542-147083491; chr2: 180125287-180126295; chr2:121974721-121975718; chr2: 122660609-122661835; chr2: 75920596-75921844;chr2: 103401408-103402401; chr2: 133843863-133844997; chr2: 180123973-180124967; chr2: 160582472-160583418; chr20: 55667282-55669174; chr22:49103355-49104388; chr3: 67113422-67114525; chr3: 74614218-74615242; chr3:5426026-5426982; chr3: 117399281-117400399; chr3: 28915343-28917419; chr3:117439560-117440548; chr3: 117237013-117239704; chr3: 137162914-137164165;chr3: 106609403-106610465; chr3: 67109328-67110455; chr3: 16048355-16049420;chr3: 117397641-117399034; chr3: 104908816-104909868; chr3: 104596179-104597355; chr4: 180058397-180059507; chr4: 180057145-180058232; chr4:27684257-27685441; chr4: 116925637-116926672; chr4: 166165437-166166501;chr4: 156168620-156169693; chr4: 18689105-18690087; chr4: 85370123-85371213;chr4: 64537927-64540013; chr4: 154982074-154983202; chr4: 130563237-130564339; chr5: 71937993-71940439; chr5: 113806007-113808129;chr5:18393819-18394795; chr5: 18465687-18466781; chr5: 123874455-123875520; chr5:123875580-123876580; chr5: 71932489-71933717; chr5: 34474174-34475918; chr5:18113286-18115121; chr5: 103808080-103809225; chr5: 144598204-144599350;chr5: 108521605-108522526; chr5: 113692453-113694380; chr5: 101678253-101679223; chr5: 87944237-87945265; chr5: 101389859-101390987; chr5:113678518-113679693; chr5: 88089287-88091255; chr5: 121797421-121798482;chr5: 87949701-87952291; chr5: 103894512-103895553; chr5: 63401153-63402160;chr6: 85922657-85923735; chr6: 104472033-104473578; chr6: 104525429-104526360; chr6: 47097062-47097998; chr6: 87968250-87969278; chr6: 16964556-16965560; chr6: 137415153-137416177; chr6: 91081186-91082896; chr6: 99746005-99747236; chr6: 99822427-99823526; chr6: 140439792-140441984; chr6:137313918-137314874; chr6: 48765024-48766063; chr6: 90890535-90891762; chr7:152925055-152926297; chr7: 12111174-12112347; chr7: 42493270-42494306; chr7:31192574-31193687; chr7: 22000950-22002150;chr7: 96785947-96787328; chr8:141715497-141716580; chr8: 26189940-26191013; chr8: 131803831-131805003;chr8: 106856111-106857217; chr8: 75975412-75977242; chr8: 115076228-115077322; chr8: 131815871-131817817; chr8: 137492454-137493716; chr9:85258346-85259505; chr9: 17904998-17907161; chr9: 78439098-78440151; chr9:16132791-16134459; chr9: 29513212-29515120; chr9: 75265233-75266237; chr9:7542343-7543943; chr9: 118169738-118170714; chr9: 71517517-71519983; chr9:7401713-7402906; chr9: 26325995-26327156; chr9: 1453442-1454897; chr9:105156511-105157922; chr9: 12425914-12426947; chrX: 68894085-68895495; chrX:20530099-20531285; chrX: 40996550-40997770; chrX: 20527023-20528155; chrX:94058277-94059471; or chrX: 138127607-138128791.; 8. An engineered cell population, wherein the engineered cells comprise: at least one exogenous GDNF gene or a functional fragment thereof.

9. The cell population of claim 8, wherein the engineered cells are human DA neurons.

10. The cell population of claim 8, wherein the human DA neurons are derived from pluripotent cells.

11. A method for preparing engineered pluripotent cells, the method comprising: - Provide a cell population containing pluripotent cells; - Introduce into a certain proportion of the pluripotent cells: i) at least one nucleic acid containing an exogenous polynucleotide sequence to be integrated into a selected endogenous locus to encode at least one GDNF gene; ii) At least one sequence-specific reagent that specifically targets the selected endogenous locus. The exogenous polynucleotide sequence is inserted into the endogenous locus through targeted gene integration.

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

13. The method of claim 11 or 12, wherein the targeted gene is integrated into the pluripotent cell via homologous recombination or NHEJ manipulation.

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

15. The method according to any one of claims 11 to 14, wherein the engineered pluripotent cells are differentiated into DA neurons to form engineered DA neurons.

16. The method of claim 15, wherein the engineered DA neurons are transplanted into a human body to form engineered DA neurons.

17. The method of claim 16, wherein the transplanted engineered DA neurons do not initially express GDNF.

18. The method of claim 16, wherein the transplanted engineered DA neurons do not express GDNF until the transplanted engineered DA neurons differentiate into mature neuronal cell types.

19. A method for preparing engineered pluripotent cells, the method comprising: - Provide a cell population containing pluripotent cells; - Introduce into a certain proportion of the aforementioned pluripotent cells: i) at least one nucleic acid containing an exogenous polynucleotide sequence to be integrated at a selected endogenous locus to encode at least one GBA gene and at least one hemizygous null SCNA gene; ii) at least one sequence-specific reagent that specifically targets the selected endogenous locus. The exogenous polynucleotide sequence is inserted into the endogenous locus through targeted gene integration.

20. The method of claim 19, wherein the engineered pluripotent cells are differentiated into DA neurons to form engineered DA neurons.

21. The method of claim 20, wherein the engineered DA neurons are transplanted into a human body to form engineered DA neurons.

22. The method of claim 21, wherein the transplanted engineered DA neurons express GBA and SCNA after transplantation.

23. The method of claim 21, wherein the transplanted engineered DA neurons do not exhibit α-synuclein accumulation.

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