Methods and compositions for inducible expression of neurotrophic factors

Abstract

To provide treating methods comprising administering a certain amount of cells to a subject afflicted by a disease or condition, and the cells.SOLUTION: Disclosed is a method of increasing glial derived neurotrophic factor (GDNF) levels in the central nervous system of an individual afflicted with a neurodegenerative disorder, comprising administering to the individual a plurality of induced pluripotent stem cell-derived neural progenitor cells (iNPCs), where the iNPCs express a genomically integrated expression cassette comprising a constitutive promoter, an inducible, bi-directional polycistronic promoter comprising a Tet response element, and a sequence encoding GDNF.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Statement regarding federally funded research This invention was made with Government support under CA202900 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] FIELD OF THEINVENTION Described herein are cells engineered to express ectopic proteins in an inducible manner. The claimed invention relates to the fields of regenerative medicine and degenerative diseases, including neurodegeneration. [Background technology]

[0003] background Since amyotrophic lateral sclerosis (ALS) is a rapidly progressive disease, constitutive trophic factor release may be sufficient without considering the long-term effects of continued administration of drugs. In contrast, many other neurological diseases with longer life spans may not require sustained growth factor secretion or delivery over long periods may be harmful. Indeed, postmortem analysis of Parkinson's disease patients who only received GDNF transiently in the caudate nucleus, caudate putamen, showed sustained neuronal sprouting, which may explain the sustained effects after cessation of GDNF delivery. Furthermore, constitutive growth factor release does not allow for discontinuation in cases of contraindication. For example, studies have reported that some patients who received GDNF developed Lhermitte symptoms. In addition, sustained GDNF expression may cause abnormal sprouting and desensitization of targeted neurons. Importantly, these effects reverted upon cessation of GDNF. Overall, there is a great need in the art for a clear rationale for the control of growth factors for these types of clinical applications, as well as compositions and methods that achieve these goals.

[0004] The combination of cell and gene therapy approaches to activate cellular niches and provide therapeutic molecules to diseased host cells is a promising new treatment approach for neurological disorders. Our group has extensively used human neural progenitor cells genetically engineered to stably produce GDNF and shown that they survive, migrate, release GDNF, and protect degenerating neurons. Importantly, these cells have been confirmed to be safe and non-tumorigenic, and as such are currently being used in the first ever FDA-approved phase 1 / 2a cell and gene therapy clinical trial for the protection of motor neurons in ALS patients.

[0005] External factors such as mifepristone, rapamycin, or tetracycline (Tet and its analog doxycycline, dox) can be used to control gene expression. Neurotrophic factors such as GDNF delivered by direct gene transfer in rodents have been controlled, but this has not yet been achieved with transplanted human neural cells engineered to release neurotrophic factors such as GDNF. Finally, the need for repeated on / off flexibility may be required, but the current standard in the field is only to induce or repress genes for a single cycle.

[0006] Herein, we describe a dox-mediated method for inducing and reversing GDNF expression in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (iNPCs) transplanted into adult mouse brain. We show that dox administration can inducibly and reversibly regulate GDNF secretion in vivo. Thus, we demonstrate that the powerful technologies of iPSC, ex vivo cell engineering, and gene regulation can be combined as a unique approach that may require controlled protein delivery to treat disorders. Summary of the Invention

[0007] Described herein are methods of treatment comprising administering to a subject suffering from a disease or condition an amount of cells, the cells expressing a therapeutic protein or peptide, and further wherein the cells, the therapeutic protein or peptide, or both, are capable of treating the disease or condition. In other embodiments, the cells are neural cells. In other embodiments, the neural cells are neural progenitor cells. In other embodiments, the neural progenitor cells are derived from induced pluripotent stem cells (iPSCs). In other embodiments, the cells express expression cassettes from one or more vectors. In other embodiments, the cells expressing expression cassettes from one or more vectors are nucleofected, transfected, or electroporated. In other embodiments, the one or more vectors comprise a piggyBac vector, a pBase vector, or both. In other embodiments, the piggyBac vector comprises at least two promoters, and at least one promoter is inducible. In other embodiments, the at least one inducible promoter is polycistronic. In other embodiments, the at least one inducible polycistronic promoter is bidirectional. In other embodiments, the expression cassette is integrated into the genome. In other embodiments, the expression cassette encodes a therapeutic protein or peptide. In other embodiments, the therapeutic protein or peptide comprises a neurotrophic factor.

[0008] In other embodiments, the neurotrophic factor comprises glial derived neurotrophic factor (GDNF). In other embodiments, the disease or condition is a neurodegenerative disease. In other embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). In other embodiments, administering an amount of cells comprises injection. In other embodiments, the method comprises administration of tetracycline, an analog or derivative thereof, including, for example, doxycycline.

[0009] Also described herein are methods that include providing a quantity of induced pluripotent stem cell (iPSC)-derived cells and introducing at least two vectors into the iPSC-derived cells. In other embodiments, the iPSC-derived cells are neural progenitor cells. In other embodiments, introducing the at least two vectors includes one or more of nucleofection, transfection, and electroporation. In other embodiments, the at least two vectors include a piggyBac vector and a pBase vector. In other embodiments, the piggyBac vector includes a constitutive promoter, an inducible promoter including a Tet-responsive element, and an expression cassette including a sequence encoding a protein or peptide, two transposon elements, and the two transposon elements flank the expression cassette. In various embodiments, the inducible promoter is a bidirectional polycistronic promoter. In other embodiments, the protein or peptide includes a neurotrophic factor. In other embodiments, the neurotrophic factor includes glial-derived neurotrophic factor (GDNF). Further described herein is a quantity of cells produced by the above-described method, wherein the iPSC-derived cells express the expression cassette integrated into the genome.

[0010] Described herein are methods that include administering to a subject suffering from a neurodegenerative disease an amount of induced pluripotent stem cell-derived neural progenitor cells (iNPCs), the cells inducibly expressing a neurotrophic factor capable of treating the disease. In other embodiments, the iNPCs express an expression cassette integrated into the genome introduced by nucleofection, the expression cassette comprising a constitutive promoter, an inducible promoter comprising a Tet-responsive element, and a sequence encoding glial-derived neurotrophic factor (GDNF). In various embodiments, the inducible promoter is a bidirectional polycistronic promoter. In other embodiments, the method includes administration of tetracycline, its analogs or derivatives, including, for example, doxycycline.

[0011] Also described herein is a quantity of neural progenitor cells that allows for inducible expression of glial derived neurotrophic factor (GDNF), comprising a quantity of induced pluripotent stem cell derived neural progenitor cells (iNPCs), the iNPCs expressing a genomically integrated expression cassette comprising a constitutive promoter, an inducible promoter comprising a Tet responsive element, and a sequence encoding GDNF. In various embodiments, the inducible promoter is a bidirectional polycistronic promoter. Further described herein is a method of treating amyotrophic lateral sclerosis (ALS) using the above-mentioned cells. In various embodiments, the genomically integrated expression cassette lacks transposon-derived sequences within 10 kb of the genomically integrated expression cassette. In various embodiments, the genomically integrated expression cassette lacks transposon-derived sequences within 1 kb of the genomically integrated expression cassette. In various embodiments, the genomically integrated expression cassette lacks virus-derived sequences within 10 kb of the genomically integrated expression cassette. In various embodiments, the genomically integrated expression cassette lacks viral-derived sequences within 1 kb of the genomically integrated expression cassette.

[0012] Also described herein is a method of increasing glial-derived neurotrophic factor (GDNF) levels in the central nervous system of an individual suffering from a neurodegenerative disorder, the method comprising administering to the individual a plurality of induced pluripotent stem cell-derived neural progenitor cells (iNPCs), the iNPCs expressing an expression cassette integrated into the genome, the expression cassette comprising a constitutive promoter, an inducible bidirectional polycistronic promoter comprising a Tet response element, and a sequence encoding GDNF. In various embodiments, the neurodegenerative disorder is selected from amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, and Alzheimer's disease. In various embodiments, the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS). In various embodiments, the neurodegenerative disorder is Parkinson's disease. In various embodiments, the neurodegenerative disorder is Alzheimer's disease. In various embodiments, the central nervous system comprises a region of the brain. In various embodiments, the region of the brain comprises the substantia nigra. In various embodiments, the region of the brain comprises the motor cortex. In various embodiments, the brain region comprises the entorhinal cortex and / or hippocampus. In various embodiments, the individual is a human. In various embodiments, the genomically integrated expression cassette is integrated into a region of the genome in which the probability of oncogenic transformation of a cell comprising the genomically integrated expression cassette is reduced. In various embodiments, the genomically integrated expression cassette is integrated into a region of the genome selected from adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) gene, and the human ortholog of the mouse Rosa26 locus. In various embodiments, the genomically integrated expression cassette is integrated into the adeno-associated virus site 1 (AAVS1). In various embodiments, the genomically integrated expression cassette is integrated into the chemokine (CC motif) receptor 5 (CCR5) gene. In various embodiments, the genomically integrated expression cassette is integrated by homologous recombination. In various embodiments, the genomically integrated expression cassette lacks sequences derived from a transposable element. In various embodiments, the expression cassette integrated into the genome is integrated by a transposon.In various embodiments, the method further comprises administering to the individual a tetracycline class antibiotic, wherein the tetracycline class antibiotic induces the transcription of GDNF mRNA in the plurality of induced pluripotent stem cell-derived neural progenitor cells. In various embodiments, the expression cassette integrated into the genome is integrated as a single copy.

[0013] Also described herein is the use of a plurality of induced pluripotent stem cell-derived neural progenitor cells (iNPCs) in a method of increasing glial-derived neurotrophic factor (GDNF) levels in the central nervous system of an individual suffering from a neurodegenerative disorder, wherein the iNPCs express a genomically integrated expression cassette comprising a constitutive promoter, an inducible bidirectional polycistronic promoter comprising a Tet-responsive element, and a sequence encoding GDNF. In various embodiments, the neurodegenerative disorder is selected from amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, and Alzheimer's disease. In various embodiments, the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS). In various embodiments, the neurodegenerative disorder is Parkinson's disease. In various embodiments, the neurodegenerative disorder is Alzheimer's disease. In various embodiments, the central nervous system comprises a region of the brain. In various embodiments, the region of the brain comprises the substantia nigra. In various embodiments, the region of the brain comprises the motor cortex. In various embodiments, the brain region comprises the entorhinal cortex and / or hippocampus. In various embodiments, the individual is a human. In various embodiments, the genomically integrated expression cassette is integrated into a region of the genome in which the probability of oncogenic transformation of a cell comprising the genomically integrated expression cassette is reduced. In various embodiments, the genomically integrated expression cassette is integrated into a region of the genome selected from adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) gene, and the human ortholog of the mouse Rosa26 locus. In various embodiments, the genomically integrated expression cassette is integrated into the adeno-associated virus site 1 (AAVS1). In various embodiments, the genomically integrated expression cassette is integrated into the chemokine (CC motif) receptor 5 (CCR5) gene. In various embodiments, the genomically integrated expression cassette is integrated by homologous recombination. In various embodiments, the genomically integrated expression cassette lacks sequences derived from a transposable element. In various embodiments, the expression cassette integrated into the genome is integrated by a transposon. In various embodiments, the expression cassette integrated into the genome is integrated as a single copy. [Brief description of the drawings]

[0014] [Figure 1] Description of the pB-RTP-Tet-GDNF / memClover-FLuc vector. (A) The pB-RTP-Tet-GDNF / memClover-FLuc plasmid is designed to stably integrate into the genome when transfected in combination with the pBase plasmid. (B) The pBase plasmid. (C) Transgenes that are constitutively expressed or expressed only in the presence of doxycycline. (D) Unstained fluorescent live imaging of human iNPCs nucleofected with pB-RTP-Tet-GDNF / memClover-FLuc and grown as neurospheres. (E) Firefly luciferase activity normalized to Renilla luciferase in iNPCs. N=4 biological replicates per condition. (F) Real-time firefly luciferase activity in live cultures of iNPCs. N=3 biological replicates per condition. Error bars represent the mean ± SEM. [Diagram 2] Doxycycline regulates GDNF expression in pB-RTP-Tet-GDNF / memClover-FLuc nucleofected iPSC-derived NPCs. Nucleofected iNPCs grown in the absence of dox (A) or presence of dox (B). Nucleofected iNPCs grown in the absence of dox (C) after dox addition and removal. (D) ELISA of cultures with 24 h incubation medium and 7 days GDNF protein accumulation of cells in (A-C). N=3 biological replicates per condition. Error bars represent mean ± SEM. **p<0.01, ***p<0.001. [Diagram 3]Reporter transgene expression is inducible and reversible in iPSC-derived NPC transplants. (A) Experimental design for nucleofection, expansion, FACS, transplantation, treatment, and postmortem analysis. (B) Weekly bioluminescence imaging of one animal from each group (animal no. 9 in group A and animal no. 3 in group B) over the 7-week in vivo experimental period. The ON / OFF button indicates whether the animal received dox in the week prior to imaging. (C) Summary of weekly bioluminescence activity of all animals (N=14). One animal (no. 13) was excluded from this analysis due to an abnormally high bioluminescence signal and no inducible transgene signal observed by postmortem immunohistochemistry. Error bars represent the mean ± SEM. [Figure 4-1] Doxycycline administration mediates GDNF expression in transplanted iPSC-derived NPCs. (A) Striatal region of animal #1 at the experimental endpoint [start off → on → off → end on] and (B) animal #7 at the experimental endpoint [start on → off → on → end off]. (C) Higher magnification image of the squared region in (A). Pink arrows indicate expression of GDNF in a region distant from TagBFP+ cells, suggesting that host cells take up secreted GDNF. (D) Higher magnification image of the squared region in (B). Note: Given the overlap (B2 / B3 and D2 / D3), Clover and GDNF signals may be background. [Figure 4-2] See description of Figure 4-1. [Diagram 5] IPSC-derived NPCs nucleofected with pB-RTP-Tet-GDNF / memClover-FLuc retain the ability to be induced upon addition of dox. 83% of BFP+ cells were also GFP+ (P5 gate) 24 hours after dox addition to cells grown in culture for 6 months post-nucleofection. Note: Cells were also exposed to two freeze / thaw cycles and frozen for a total of 10 months during this period. [Figure 6] GDNF DAB staining of animals shows increased signal in animals terminated on dox. Related to FIG. [Figure 7] GDNF, SC121 (human cytoplasmic), and SC101 (human nuclear) staining of the striatum / corpus callosum regions of all animals used in the study show increased GDNF signal in animals that completed the study on-dox. These data were used for blind grading of GDNF signal shown in Table 2 (RH=right hemisphere, LH=left hemisphere). [Figure 8] Vector map of AAVS1-teton-hGDNF. The HA-L and HA-R arms are shown, which are homologous recombination sequences that can be used to target genomic safe harbors such as AAVS. [Figure 9] Scheme for assessment of inducible GDNF expression. [Figure 10] pDonor-Teton3g-2a-TagBFP-V5-nls-p2a-puroR WPRE_isolate mpclover-2a-luc2pest-2a-gdnf wpre vector map. [Figure 11](A) Schematic of AAVS1 targeting of the endogenous locus between exons 1 and 2 of the human PPP1R12C gene. First, a recipient "landing site" consisting of a splice acceptor-driven reporter / selection cassette (TagBFP2 and PuroR for fluorescence and antibiotic selection) linked to the upstream PPP1R12C, and a constitutive CAG promoter-driven td-Tomato red fluorescent cistron flanked by LoxP and FRT sites, was stably integrated. Subsequently, upon stable selection for these reporters, the cells were lipofected with a plasmid expressing FlpO and Cre, the donor plasmid contains the LoxP and FRT-flanked selection / reporter cassette, and lipofected with a dox-inducible mpClover / Luc2p / GDNF cistron-containing plasmid. (B) Transmitted light and green fluorescent imaging of cells in the absence and presence of doxycycline shows inducibility of mpClover 24 hours after dox addition, and the absence of GFP "leakage" in the absence of dox. Additionally, the cells were examined approximately 1 week after selection to enrich for transfected cells and 2 weeks after transfection to ensure that the mpClover population was a stable transfectant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise noted, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006), and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 4.th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provides those of skill in the art with a general guide to many of the terms used in this application.

[0016] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0017] The term "control elements" collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of the coding sequence in the recipient cell. Not all of these control elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0018] The term "promoter region" is used herein in its conventional sense to refer to a nucleotide region that contains DNA regulatory sequences derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence.

[0019] "Operably linked" refers to the arrangement of elements such that the components so described are configured to perform their normal function. Thus, control elements operably linked to a coding sequence are capable of affecting the expression of the coding sequence. Control elements need not be contiguous with the coding sequence, so long as they function to induce expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0020] Using stem cell-derived neural progenitor cells to deliver trophic factors to the brain is a powerful way to circumvent the blood-brain barrier. Using ex vivo genetically modified cells to deliver various growth factors to the site of injury has been shown to support host neurons in disease models of amyotrophic lateral sclerosis (ALS) as well as Parkinson's, Huntington's, and Alzheimer's. In parallel, delivery of glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinson's disease patients and is currently being tested in phase 1 / 2a clinical trials for ALS patients. To fully exploit the benefits of trophic factors and avoid potential undesirable effects of cell-delivered trophic factors, control of growth factor secretion is a promising avenue for several neurodegenerative diseases. Delivery of trophic factors over long periods of time is not dose-adjustable or is stopped if side effects occur, as gene expression and activation of downstream signaling are closely linked processes. Lack of control over the timing and magnitude of gene expression can limit the efficacy of treatment and result in unintended cellular effects.

[0021] Towards these ends, the tetracycline (Tet) regulatory system has been used to control gene expression temporally and spatially in a variety of methodologies. This includes the bacterial Tet transactivator (tTA), which represses gene expression downstream of a Tet-regulated promoter in the presence of the Tet analogue doxycycline (dox). In addition to this "Tet-Off" system, the "Tet-On" system uses reverse tTA (rtTA) to activate transgene expression in the presence of dox. The use of tTA and rtTA variants in neural stem cell populations has not been explored.

[0022] Described herein is a stably integrated third generation doxycycline-regulated vector that allows for inducible and reversible expression of therapeutic molecules. Human iPSC-derived neural progenitor cells were stably transfected with the vector, expanded, and transplanted into adult mouse brain. We observed that the addition and withdrawal of doxycycline resulted in GDNF expression that could be induced and reversed multiple times, indicating that doxycycline can penetrate grafts in vivo to control transgene expression. Our findings provide a proof of concept for combining gene and stem cell therapy for effective regulation of ectopic protein expression in transplanted cells.

[0023] Described herein are methods of treatment that include administering to a subject suffering from a disease or condition an amount of cells, the cells expressing a therapeutic protein or peptide, and the cells, the therapeutic protein or peptide, or both, are capable of treating the disease or condition. In other embodiments, the cells are neural cells. In other embodiments, the neural cells are neural progenitor cells. In other embodiments, the neural progenitor cells are derived from induced pluripotent stem cells (iPSCs). In various embodiments, the neural progenitor cells are generated from iPSC colonies by culturing in neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (called EZ spheres) can be grown for long periods of time using a chopping method that maintains cell-cell contact. In various embodiments, the EZ spheres are removed from EGF / FGF cultured with retinoic acid RA in neural induction. Techniques relating to neural progenitor cells derived from iPSCs are described in Sareen et al. "Human neural progenitor cells generated from induced pluripotent stem cells can survive, migrate, and integrate in the rodent spinal cord" J Comp Neurol. 2014 Aug 15;522(12):2707-2728 and Ebert et al., "EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs" Stem Cell Res. 2013 May;10(3):417-427, which are incorporated by reference in their entireties. In other embodiments, iPSCs include somatic cells such as fibroblasts and blood that have been reprogrammed according to the methods described in U.S. Publication No. 2017 / 0362574, which is incorporated by reference in its entirety.

[0024] In other embodiments, the cells express expression cassettes from one or more vectors. In other embodiments, the cells expressing expression cassettes from one or more vectors are nucleofected, transfected, or electroporated, or by other gene delivery techniques known in the art. In other embodiments, the one or more vectors include a piggyBac vector, a pBase vector, or both. In other embodiments, the piggyBac vector includes at least two promoters, and at least one promoter is inducible. In other embodiments, the at least one inducible promoter is polycistronic. In other embodiments, the at least one inducible polycistronic promoter is bidirectional. In other embodiments, the expression cassette is integrated into the genome. In other embodiments, the expression cassette encodes a therapeutic protein or peptide. In other embodiments, the therapeutic protein or peptide includes a neurotrophic factor.

[0025] In various embodiments, the one or more vectors include a vector having a gene expression cassette flanked by two transposon elements. In various embodiments, the two transposon elements include piggyBac terminal repeats (PB TR). In various embodiments, the vector includes a constitutive promoter, which includes a CMV / chicken β-actin (also known as CAG) promoter. In various embodiments, the vector includes an includable bidirectional promoter, which includes a TRE-Bi promoter. In various embodiments, the constitutive promoter is operably linked to a Tet response element. In various embodiments, the "Tet-On" element includes, for example, rTA. In other embodiments, the rTA includes rtTA-V10. In various embodiments, the constitutive promoter is operably linked to a selection element, including, for example, neomycin or puromycin. In various embodiments, the inducible bidirectional promoter is polycistronic. In various embodiments, the inducible bidirectional promoter is operably linked to a first, second, or third, or more cistron elements. In various embodiments, the first, second, or third or more cistrons comprise a transgene. In various embodiments, the transgene is followed by one or more post-transcriptional factors. In various embodiments, the one or more post-transcriptional factors comprise a Woodchuck Hepatitis Virus Post-Transcriptional Factor (WPRE). In various embodiments, the transgene is followed by one or more polyA tails, including, for example, rabbit β-globin polyA. In various embodiments, the transgene is a neurotrophic factor. In various embodiments, the neurotrophic factor comprises glial derived neurotrophic factor (GDNF). Additional information can be found in PCT Publication No. WO2017 / 131926 and Akhtar et al. "A Transposon-Mediated System for Flexible Control of Transgene Expression in Stem and Progenitor-Derived Lineages" Stem Cell Reports. 2015 Mar 10;4(3):323-331, which are incorporated herein by reference in their entireties.

[0026] In other embodiments, the one or more vectors include vectors encoding recombinases, including VCre (Vlox and derivatives), SCre (Slox and derivatives), Dre (Rox and derivatives), and phiC31 (attb) or other recombinases known in the art.

[0027] In other embodiments, the neurotrophic factor comprises glial derived neurotrophic factor (GDNF). In other embodiments, the disease or condition is a neurodegenerative disease. In other embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). In other embodiments, administering an amount of cells comprises injection. In other embodiments, the method comprises administration of tetracycline, an analog or derivative thereof, including, for example, doxycycline.

[0028] In various embodiments, the vector comprises at least one homologous recombination sequence. In other embodiments, the homologous recombination sequence comprises a sequence capable of targeting a genomic safe harbor. In other embodiments, the genomic safe harbor is one of the following: adeno-associated virus site 1 (AAVS1), chemokine (CC motif) receptor 5 (CCR5) gene, human orthologue of mouse Rosa26 locus. Exemplary sequences of one of the above-mentioned vectors are SEQ ID NO:2 and SEQ ID NO:3.

[0029] Described herein are methods that include providing a quantity of induced pluripotent stem cell (iPSC)-derived cells and introducing at least two vectors into the iPSC-derived cells. In other embodiments, the iPSC-derived cells are neural progenitor cells. In other embodiments, introducing the at least two vectors includes one or more of nucleofection, transfection, and electroporation. In other embodiments, the at least two vectors include a piggyBac vector and a pBase vector. In other embodiments, the piggyBac vector includes a constitutive promoter, an inducible bidirectional polycistronic promoter including a Tet response element, and an expression cassette including a sequence encoding a protein or peptide, two transposon elements, and the two transposon elements flank the expression cassette. In other embodiments, the protein or peptide includes a neurotrophic factor. In other embodiments, the neurotrophic factor includes glial derived neurotrophic factor (GDNF). In various embodiments, the vector includes at least one homologous recombination sequence. In various embodiments, the vector and / or cell comprising the vector lacks transposable elements and / or transposon derived sequences within 500 bp, 1 kb, 2 kb, 5 kb, or 10 kb of the expression cassette. In various embodiments, the vector and / or cell comprising the vector lacks virus derived sequences within 500 bp, 1 kb, 2 kb, 5 kb, or 10 kb of the expression cassette. In various embodiments, the cell comprises one or two copies of the vector integrated into the genome. In various embodiments, the cell comprises the vector integrated into the genome as a single copy.

[0030] Also described herein is a quantity of cells produced by the above-mentioned method, wherein the iPSC-derived cells express the expression cassette integrated into the genome. For example, a quantity of neural progenitor cells that allows inducible expression of glial-derived neurotrophic factor (GDNF) is produced by a method comprising providing a quantity of induced pluripotent stem cell-derived neural progenitor cells (iNPCs) and introducing a piggyBac vector and a pBase vector into the iNPC-derived cells, wherein the piggyBac vector comprises a constitutive promoter, an inducible bidirectional polycistronic promoter comprising a Tet-responsive element, and a sequence encoding GDNF. In various embodiments, the vector comprises at least one homologous recombination sequence.

[0031] Further described herein are methods of treating degenerative diseases and / or conditions using the above-described cells, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

[0032] Described herein are methods that include administering to a subject suffering from a neurodegenerative disease an amount of induced pluripotent stem cell derived neural progenitor cells (iNPCs), the cells inducibly expressing a neurotrophic factor capable of treating the disease. In other embodiments, the iNPCs express a genomically integrated expression cassette introduced by nucleofection, the expression cassette comprising a constitutive promoter, an inducible bidirectional polycistronic promoter comprising a Tet response element, and a sequence encoding glial derived neurotrophic factor (GDNF). In various embodiments, the vector comprises at least one homologous recombination sequence. In other embodiments, the method comprises administration of tetracycline, an analog or derivative thereof.

[0033] Also described herein is a quantity of neural progenitor cells that allows for inducible expression of glial derived neurotrophic factor (GDNF), the neural progenitor cells comprising a bidirectional polycistronic promoter that includes a Tet response element, and an expression cassette integrated into the genome that includes a sequence encoding GDNF. Further described herein are methods of treating degenerative diseases and / or conditions using the above-described cells, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

[0034] Described herein is a vector comprising a constitutive promoter, an inducible bidirectional promoter comprising a Tet response element, and a gene expression cassette comprising a sequence encoding a protein or peptide. In various embodiments, the gene expression cassette is flanked by two transposon elements. In various embodiments, the two transposon elements comprise piggyBac terminal repeats (PB TR). In various embodiments, the two transposon elements comprise loxP and flippase recognition targets, or other transposon elements known in the art. In various embodiments, the constitutive promoter comprises a CMV / chicken β-actin (also known as CAG) promoter. In various embodiments, the includable bidirectional promoter comprises a TRE-Bi promoter. In various embodiments, the constitutive promoter is operably linked to a Tet response element. In various embodiments, the Tet response element is a "Tet-Off" element, e.g., comprising tTA, or a "Tet-On" element, e.g., comprising rTA. In other embodiments, the rTA comprises rtTA-V10. In various embodiments, the constitutive promoter is operably linked to a selection factor, including, for example, neomycin or puromycin. In various embodiments, in embodiments, the inducible bidirectional promoter is polycistronic. In various embodiments, the inducible bidirectional promoter is operably linked to factors of a first, second, or third, or more cistrons. In various embodiments, the first, second, or third, or more cistrons include a transgene. In various embodiments, the transgene is followed by one or more post-transcriptional factors. In various embodiments, the one or more post-transcriptional factors include Woodchuck Hepatitis Virus Post-Transcriptional Factor (WPRE). In various embodiments, the transgene is followed by one or more polyA tails, including, for example, rabbit β-globin polyA. In various embodiments, the transgene is a neurotrophic factor. In various embodiments, the neurotrophic factor includes glial derived neurotrophic factor (GDNF). In various embodiments, the vector includes one or more factors that facilitate targeting of a safe landing site, including AAVS1.In various factors, one or more insulator factors around the inducible cassette attenuate potential silencing during cell differentiation. In various embodiments, the expression cassette comprises one or more sub-cassettes, each sub-cassette comprising 1) a promoter, 2) a transgene, and 3) a polyA transcription termination element. In various embodiments, the expression cassette comprising one or more sub-cassettes comprises a constitutive sub-cassette and an inducible sub-cassette. For example, the constitutive sub-cassette comprises a constitutive promoter that expresses rTA transactivator, and the inducible sub-cassette comprises an inducible promoter that expresses a neurotrophic factor such as GDNF and optionally one or more reporter proteins.

[0035] An example of the above vector is pB-RTP-Tet-GDNF / memClover-FLuc[ p iggy B ac- R everse transactivator / T agBFP2nls / P acR- Tet inducible- GDNF / mem Brane Clove r- F irefly Luciferase][SEQ ID NO:1], which is shown in FIG. 1A. Here, the vector contains two promoters, a constitutively active CMV / chicken β-actin (aka CAG) promoter and an inducible bidirectional TRE-Bi promoter. The CAG promoter drives the constitutive expression of the rtTA-V10 (aka Tet-On) transactivator, TagBFP2-V5nls (enhanced blue fluorescent protein with V5 tag and nuclear localization sequence), and a puromycin resistance gene. The tandem transgenes are separated by a self-cleaving peptide linker (P2A). Here, the addition of doxycycline, a tetracycline analog or derivative, allows the rtTA-V10 transactivator to bind to the TRE-Bi promoter and catalyze the transcription of the downstream transgene. The first cistron of the TRE-Bi promoter has a myristoylated and palmitoylated (MyrPalm) Clover reporter (mpClover) followed by an unstable firefly luciferase (Luc2P). The second cistron downstream of the inducible TRE-Bi promoter may encode a neurotrophic factor such as GDNF followed by a woodchuck hepatitis virus post-transcriptional factor (WPRE) for increased gene expression. Rabbit β-globin polyA was placed downstream of each factor to stop transcription and prevent spurious transgene expression. The pB-RTP-Tet-GDNF / memClover-FLuc vector can be transfected together with the pBase plasmid to promote stable genomic integration. Another example of the above-mentioned vector is pDonor-Teton3g-2a-TagBFP-V5-nls-p2a-puroR WPRE_sequestered mpclover-2a-luc2pest-2a-gdnf wpre shown in FIG. 10.

[0036] In various embodiments, the vector comprises at least one homologous recombination sequence. In other embodiments, the homologous recombination sequence comprises a sequence capable of targeting a genomic safe harbor. In other embodiments, the genomic safe harbor is one of the following: adeno-associated virus site 1 (AAVS1), chemokine (CC motif) receptor 5 (CCR5) gene, human orthologue of mouse Rosa26 locus. Exemplary sequences of one of the above-mentioned vectors are SEQ ID NO:2 and SEQ ID NO:3.

[0037] Methods of treatment are described herein that include administering an amount of cells to a subject suffering from a disease or condition, the cells expressing a therapeutic protein or peptide, and further, the cells, the therapeutic protein or peptide, or both, are capable of treating the disease or condition. In each and all of the above-mentioned embodiments of the method, the cells are neural cells. In each and all of the above-mentioned embodiments of the method, the neural cells are neural progenitor cells. In each and all of the above-mentioned embodiments of the method, the neural progenitor cells are derived from induced pluripotent stem cells (iPSCs). In each and all of the above-mentioned embodiments of the method, the cells express expression cassettes from one or more vectors. In each and all of the above-mentioned embodiments of the method, the cells expressing expression cassettes from one or more vectors are nucleofected, transfected, or electroporated. In each and all of the above-mentioned embodiments of the method, the one or more vectors include piggyBac vectors, pBase vectors, or both. In each and all of the above-mentioned embodiments of the method, the piggyBac vector comprises at least two promoters, and at least one promoter is inducible. In each and all of the above-mentioned embodiments of the method, the at least one inducible promoter is polycistronic. In each and all of the above-mentioned embodiments of the method, the at least one inducible polycistronic promoter is bidirectional. In each and all of the above-mentioned embodiments of the method, the expression cassette is integrated into the genome. In each and all of the above-mentioned embodiments of the method, the expression cassette encodes a therapeutic protein or peptide. In each and all of the above-mentioned embodiments of the method, the therapeutic protein or peptide comprises a neurotrophic factor. In each and all of the above-mentioned embodiments of the method, the neurotrophic factor comprises glial-derived neurotrophic factor (GDNF). In each and all of the above-mentioned embodiments of the method, the disease or condition is a neurodegenerative disease. In each and all of the above-mentioned embodiments of the method, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).In each and every one of the above embodiments of the method, administering an amount of cells comprises injection.In each and every one of the above embodiments of the method, the method comprises administration of a tetracycline, an analog or a derivative thereof.

[0038] Described herein are methods that include providing a quantity of induced pluripotent stem cell (iPSC)-derived cells and introducing at least two vectors into the iPSC-derived cells. In each and all of the above-mentioned embodiments of the method, the iPSC-derived cells are neural progenitor cells. In each and all of the above-mentioned embodiments of the method, introducing at least two vectors includes one or more of nucleofection, transfection, and electroporation. In each and all of the above-mentioned embodiments of the method, the at least two vectors include a piggyBac vector and a pBase vector. In each and all of the above-mentioned embodiments of the method, the piggyBac vector includes a constitutive promoter, an inducible bidirectional polycistronic promoter including a Tet response element, and an expression cassette including a sequence encoding a protein or peptide, two transposon elements, and the two transposon elements flank the expression cassette. In each and all of the above-mentioned embodiments of the method, the protein or peptide includes a neurotrophic factor. In each and all of the above-mentioned embodiments of the method, the neurotrophic factor includes glial-derived neurotrophic factor (GDNF). In each and every one of the above-mentioned embodiments of the method, the piggyBac vector comprises a homologous recombination sequence. In each and every one of the above-mentioned embodiments of the method, the homologous recombination sequence comprises a sequence capable of targeting a genomic safe harbor. In each and every one of the above-mentioned embodiments of the method, the genomic safe harbor is one of the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) gene, and the human orthologue of the mouse Rosa26 locus.

[0039] This further includes a quantity of cells produced by each and all of the above described embodiments of the method, wherein the iPSC-derived cells express the expression cassette integrated into the genome.

[0040] Also described herein are methods that include administering to a subject suffering from a neurodegenerative disease an amount of induced pluripotent stem cell-derived neural progenitor cells (iNPCs), the cells inducibly expressing a neurotrophic factor capable of treating the disease. In each and every of the above-mentioned embodiments of the method, the iNPCs express an expression cassette induced by nucleofection and integrated into the genome, the expression cassette comprising a constitutive promoter, an inducible bidirectional polycistronic promoter comprising a Tet-responsive element, and a sequence encoding glial-derived neurotrophic factor (GDNF). In each and every of the above-mentioned embodiments of the method, the method comprises administration of tetracycline, an analogue or derivative thereof. EXAMPLES

[0041] Non-limiting examples of the claimed invention are described herein.

[0042] Example 1 Dox supplementation of iPSCs and iNPCs in cell culture or animals For all experiments, Dox solutions were kept at 4 °C protected from light. For cell cultures, dox (Clontech 631311) was added to the medium at 100 ng / ml. For in vivo studies, animals were administered dox (15 μg dox / g body weight) every 3–4 days by oral gavage (e.g., 60 μl for a 20 g animal) using a soft-tipped feeding needle (Instech FTP-20-30, Plymouth Meeting, PA) attached to a 1 ml syringe (BD 309659, Franklin Lakes, NJ).

[0043] For example, reprogramming of somatic cells to generate iPSCs and subsequent generation of iNPCs has been described, including Sareen et al. "Human neural progenitor cells generated from induced pluripotent stem cells can survive, migrate, and integrate in the rodent spinal cord" J Comp Neurol. 2014 Aug 15;522(12):2707-2728, which is incorporated herein by reference in its entirety.

[0044] In summary, for reprogramming and iPSC generation, cells such as fibroblasts are reprogrammed into virus-free iPSC lines using the Amaxa Human Dermal Fibroblast Nucleofector Kit expressing episomal plasmids (Addgene) carrying six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Exogenously introduced genes are not integrated, but instead expressed episomally in a transient manner. In summary, fibroblasts (0.8×106 cells per nucleofection) are harvested, centrifuged at 200g for 5 minutes, carefully resuspended in Nucleofector® solution (VPD-1001, Lonza), and the U-023 program is applied. During reprogramming, all cultures are maintained in standard oxygen conditions (5% O2), which further increases the efficiency of iPSC generation. The medium was maintained for 48 h and gradually replaced with chemically defined mTeSR®1 medium containing small molecules to enhance reprogramming efficiency. The small molecules used were: 1) sodium butyrate (0.5 mM), 2) glycogen synthase kinase 3β inhibitor of the Wnt / β-catenin signaling pathway (CHIR99021, 3 μM), 3) MEK pathway inhibitor (PD 0325901, 0.5 μM), 4) selective inhibitor of TGF-β type I receptor ALK5 kinase, type I activin / nodal receptor ALK4, and type I nodal receptor ALK7 (A83-01, 0.5 μM). Colonies with ES / iPSC-like morphology appeared after 25–31 days. Subsequently, colonies with the best morphology were transferred onto feeder-independent 6BD Matrigel™ matrix and maintained in mTeSR®1 medium. iPSC clones were further expanded and cryopreserved.

[0045] EZ spheres generated from iPSCs can be differentiated into cultures of neural progenitor cells in suspension (iNPCsSU) with astrogliogenic predisposition. After EGF / FGF2 / heparin withdrawal, EZ spheres were caudalized using retinoic acid RA (0.5 μM) in neural induction medium (NIM) (DMEM / F12, 1% NEAA, 1% N2, heparin 2 μg / ml, Sigma). This medium was replaced every 2 days for the next 11 days, after which a stable population of iNPCsSU was reintroduced into StemHi E / F / H for expansion by weekly chopping (similar to EZ spheres). iNPCsSU maintain their proliferation potential and astrogliogenic propensity for 26–30 passages and can be efficiently cryopreserved. Additionally, iNPCs grown as adherent cultures and in adherent form, termed iNPCsAD, were generated by Accutase dissociation of EZ spheres for plating on growth factor reduced Matrigel (Corning) at a density of 10,000 cells / cm2 in StemHi E / F / H and passaged weekly using TrypLE (Life Technologies). Towards differentiation into astrocytes, iNPCsSU were dissociated into single cells with Accutase (BD Biosciences) or iNPCsAD harvested with TrypLE were plated on poly-l-ornithine / Matrigel coated glass coverslips at 25000 cells / cm2 in NIM for 7-21 days. Additional information can be found in Ebert et al., “EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs” Stem Cell Res. 2013 May;10(3):417-427.

[0046] Example 2 transplant Nucleofected iNPCs, which underwent two rounds of FACS for BFP, were cultured in 6-month-old NOD-SCID mice (Nod.cb17-Prkdc scid100,000 cells / μl in each hemisphere). A subset of cells was treated with dox 3 days prior to transplantation. For transplantation, cells were dissociated and diluted in conditioned medium to a final concentration of 100,000 cells / μl and kept on ice. Animals were anesthetized with isoflurane and placed in a stereotaxic frame. Cells were injected both 0.7 mm rostral to the bregma and + / - 2.0 mm lateral. A 5 μl Hamilton microsyringe filled with PBS was filled with 2 μl of cell solution and inserted to a depth of -4.00 mm. The microsyringe was then raised to -3.50 mm and cell solution was injected at 1 μl / min. After cell injection, the microsyringe was raised to -3.00 mm and held for 2 minutes and gradually withdrawn. Animals were checked daily.

[0047] Example 3 New vectors providing regulated expression of reporter genes and GDNF The Tet-On system relies on the constitutive expression of the rtTA-V10 transactivator and the dox-responsive TRE-Bi (Tet-responsive element bidirectional) promoter. We observed that when the transactivator and the inducible promoter were expressed on different plasmids and co-transfected into cells, the cells drifted during growth and were selected for a double transgenic population, resulting in non-induced cells (data not shown). Incorporating these factors and a reporter gene into a single system required a large vector, which we newly created, "pB-RTP-Tet-GDNF / memClover-FLuc" [ p iggy B ac- R everse transactivator / T agBFP2nls / P acR- Tet inducible- GDNF / mem Brane Clove r- F irefly LucThis was achieved with a vector called pBase enzyme [pBaseR] (Figure 1A). The vector is flanked by piggyBac terminal repeats (PB TR) (Figure 1A1), which allow stable genomic integration when the pBase enzyme is transiently expressed.

[0048] The vector has two promoters: a constitutively active CMV / chicken β-actin (aka CAG) promoter and an inducible bidirectional TRE-Bi promoter. The CAG promoter (Figure 1A2) drives the constitutive expression of the rtTA-V10 (aka Tet-On) transactivator (Figure 1A3), TagBFP2-V5nls (enhanced blue fluorescent protein with V5 tag and nuclear localization sequence) (Figure 1A4), and a puromycin resistance gene (Figure 1A5). The tandem transgenes are separated by a self-cleaving peptide linker (P2A). Addition of dox to the system (Figure 1A6) causes the rtTA-V10 transactivator to undergo a conformational change (Figure 1A7), which allows it to bind to the TRE-Bi promoter (Figure 1A8), catalyzing the transcription of the downstream transgene. Withdrawal of dox reverses the induced gene expression. The first cistron of the TRE-Bi promoter encodes a myristoylated and palmitoylated (MyrPalm) Clover reporter (mpClover) (Figure 1A9), followed by an unstable firefly luciferase (Luc2P) (Figure 1A 10 ), where Clover is a green fluorescent protein and the MyrPalm sequence localizes to the cell membrane. A membrane fluorescent reporter was chosen to avoid visual overlap with the perinuclear localization of GDNF. An unstable firefly luciferase (Luc2P), with a half-life several times shorter than wild-type luciferase, was used for analysis of gene expression in living animals over time. A second cistron downstream of the inducible TRE-Bi promoter encodes GDNF (Figure 1A) for increased gene expression. 11 ), followed by the woodchuck hepatitis virus post-transcriptional element (WPRE) ( Fig. 1A 12). A well-characterized rabbit β-globin polyA was placed downstream of each factor to silence transcription and prevent spurious transgene expression. The pB-RTP-Tet-GDNF / memClover-FLuc vector was designed to be co-transfected with the pBase plasmid (Fig. 1B) to promote stable genomic integration (Fig. 1B1). In summary, the presence or absence of dox affects the expression of the inducible transgenes (Clover, luciferase, and GDNF), while the rtTA-v10 transactivator, TagBFP-v5-nls, and puromycin resistance genes are constitutively expressed (Fig. 1C). Additional information can be found in PCT Publication No. WO2017 / 131926 and Akhtar et al. “A Transposon-Mediated System for Flexible Control of Transgene Expression in Stem and Progenitor-Derived Lineages” Stem Cell Reports. 2015 Mar 10;4(3):323-331, which are incorporated by reference in their entireties.

[0049] Example 4 Immunocytochemistry of nucleofected iPSC-derived NPCs To promote cell attachment, coverslips were coated with polyornithine (Sigma, P4638) and subsequently treated with 25 μl of laminin (50 μg / ml, Sigma-Aldrich L2020) for 1 h at 37 °C. The coating was then removed. Nucleofected cells were removed from 6-well plates by incubating in TrypLE for 5 min. An equal volume of medium was added to neutralize the enzymatic reaction, and then dissociated cells were counted, pelleted at 150 rcf, resuspended in medium at 1,000 cells / μl, and 25 μl of cells were pipetted onto the laminin-coated area of ​​the coverslip. To allow cell attachment, after 4–6 h of incubation at 37 °C, wells were gently filled with 500 μl of growth medium. Half the volume of medium was replaced every 24 h.

[0050] Immunocytochemistry was performed as described above. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 12 min. The PFA solution was then removed and washed three times with phosphate-buffered saline (PBS), each incubated for 5 min. Cells were then incubated for at least 12 h at 4°C in primary antibodies [concentrations listed in Table 1] diluted in PBS containing 0.3% Triton (PBS-T) and 3% normal donkey serum (NDS) on a gentle rocker, followed by three 5-min PBS washes at room temperature. Secondary antibodies (conjugated with Alexa405, FITC, Alexa488, Dylight488, Alexa555, Dylight549, Alexa647, or Dylight649, Jackson Immunoresearch, West Grove, PA) were diluted in PBS-T at a 1:1000 dilution and incubated for 1 h at room temperature on a gentle rocker. Coverslips were then washed with PBS and mounted onto glass slides using antifade mounting gel medium (Invitrogen ProLong Gold, P10144).

[0051] Table 1. Antibodies used in this study TIFF2024073654000002.tif51153

[0052] Example 5 ELISA The medium used for the ELISA consisted of DMEM:F12 (70:30) without EGF and FGF, supplemented with 1% antibiotic-antimycotic, and 2% B27 without vitamin A. Before adding medium during the 24 h or 7 day incubation period, old medium was aspirated and wells were washed twice with sterile PBS. Collected medium was stored at -80°C. GDNF ELISA was performed according to the manufacturer's instructions (R&D Systems DY212, Minneapolis, MN).

[0053] Example 6 Imaging After immunocytochemistry and immunohistochemistry fluorescent staining, confocal images were collected on a Nikon A1R inverted laser scanning confocal microscope with appropriate settings to image colors sequentially and avoid signal crosstalk. Exposure and saturation measurements were utilized to capture the maximum dynamic range. Typically, after exposure was set, identical settings were reused for subsequent samples within a group. ND2 image files were first imported into ImageJ to manipulate confocal Z-stacks or to separate individual channels from a single Z-slice for subsequent editing in Adobe Photoshop CS6. Image curves were adjusted and cropped for consistency of dynamic range and exposure in Photoshop CS6, and then imported into Adobe Illustrator CS6 to prepare the final images.

[0054] Example 7 GDNF scoring of transplanted tissues Immunostained sections were photographed using a Leica DM200 LED microscope, and images were scored based on the intensity of DAB GDNF staining by an observer blinded to experimental condition (dox vs. no dox).

[0055] Example 8 The production of the reporter protein is reversibly regulated in vitro. To evaluate the dox-regulated system in therapeutically relevant cells, human iNPCs were nucleofected with pB-RTP-Tet-GDNF / memClover-FLuc and pBase vectors. Nucleofected cells were grown as floating spheres (called EZ spheres) that can be easily maintained and rapidly expanded. In the absence of dox, the spheres were induced by BFP + Within 24 hours of dox addition, the spheres constitutively expressed BFP + and GFP + Both the TRE-Bi promoter and the IL-11 promoter were expressed, indicating that the TRE-Bi promoter was responsive to dox (FIG. 1D).

[0056] To quantitatively assess reporter expression from the TRE-Bi promoter, iNPCs were nucleofected, as described above, with an Ef1-Renilla plasmid constitutively expressing Renilla luciferase for protein normalization. Dual luciferase assays revealed that cellular firefly luciferase activity (normalized to Renilla) represented a ∼1300-fold increase after dox treatment at 24 h, which returned to near basal levels upon dox withdrawal (Figure 1E). To assess the kinetics of the TRE-Bi-inducible promoter, firefly luciferase activity was quantified in live cultures of nucleofected iNPCs grown in the presence of beetle luciferin (Figure 1F). Results showed that firefly bioluminescence rapidly increased after dox addition at time 0 and gradually decreased after dox removal (at 48 h). To assess whether the TRE-Bi promoter could be reversibly induced, hourly images of nucleofected iNPCs were taken using the IncuCyte S3 Live-Cell Analysis system. Results showed that clover expression could be induced, reverted, and re-induced by adding, removing, and re-adding dox to the cultures. Given the need to expand and preserve clinically relevant cell products, we assessed whether the TRE-Bi promoter maintained a functional response following extensive cell expansion and freezing. Flow analysis demonstrated that constitutively induced BFP + It was found that 83% of the cells were induced to express GFP (Fig. 5 ), indicating that the TRE-Bi promoter remained functional.

[0057] Example 9 GDNF production is reversibly regulated in vitro. To ensure that our system can control therapeutically relevant molecules, we next assessed the production of GDNF in nucleofected iNPCs. Immunofluorescence revealed that GDNF was not visible in cells in the absence of dox. This confirms our previous report that wild-type cells do not produce GDNF. The reporter proteins Clover and luciferase were also not produced in the absence of dox, whereas BFP was produced under a constitutive promoter (Figure 2A). In the presence of dox, GDNF as well as Clover and luciferase were detected, in addition to maintaining BFP production (Figure 2B). When dox was subsequently removed, all GDNF and reporter gene production ceased, but BFP was maintained (Figure 2C).

[0058] To quantify the regulation of GDNF, we used ELISA on cells grown in the presence of dox (on), in the absence of dox (off), in the presence of dox followed by the absence of dox (on → off), and in the presence of dox for an extended period of time (on → on), without changing the medium (Figure 2D). Again, cells did not produce appreciable GDNF in the absence of dox. In contrast, cells produced robust levels of GDNF in the presence of dox after only 24 hours, which accumulated over 7 days of continued dox treatment. Importantly, GDNF can be reversibly turned on or returned to baseline levels based on the presence or absence of dox. Normalizing the ELISA results using stereological counts of GDNF-positive cells revealed that dox-treated cultures produced 7.34x10 GDNF-positive cells per hour. -4 It was shown that they secreted picograms of GDNF. Taken together, the demonstration that a protein can be efficiently induced and reversed in vitro lays the groundwork for protein regulation in cells following transplantation.

[0059] Example 10 Upon iNPC transplantation into the adult brain, reporter transgene expression is inducible and reversible. To investigate the ability of this system to control protein expression in vivo, nucleofected iNPCs were expanded and transplanted into adult NOD-SCID mouse brains (Figure 3A). During in vitro expansion, cells underwent two FACS rounds for nuclear BFP, and the parental cell population had a BFP of 15.6%. + These were then expanded as neurospheres for 7 weeks and selected a second time for BFP, which contained 32.4% BFP. + After 3 weeks of the second sorting, the cells were divided into two groups, one group of cells received dox for 3 days in culture and before transplantation, and the other group of cells did not receive dox. 200,000 iNPCs were transplanted into each striatal hemisphere in 6-month-old NOD-SCID mice (n=14), 10 animals received cells pretreated with dox and 4 animals received untreated cells. Animals did not receive dox before transplantation.

[0060] One day after transplantation, all 10 animals implanted with dox-treated cells emitted luciferase signals above background, which were calculated based on the signal in the posterior region of the animals distal to the CNS (Figure 3B-C, week 0). To determine whether the implanted cells could be subsequently turned "on" and "off" multiple times by administration and withdrawal of dox, animals were divided into two groups and cycled as follows: on → off → on → off → on (group A) or off → on → off → on (group B). Weekly bioluminescence imaging showed that dox effectively controlled luciferase protein in vivo in both representative animals (animals that started the study on dox and ended off dox, as well as animals that started off dox and ended on dox) (Figure 3B). Quantification of luciferase activity at weekly imaging sessions confirmed both the increase in luciferase activity in dox-treated animals and the inducibility of gene expression over time (Figure 3C). Taken together, this provides in vivo proof of principle for dox-inducible gene expression.

[0061] Example 11 In iNPC transplants, GDNF expression is inducible and reversible. To confirm the control of GDNF expression in the transplanted cells, animals were euthanized after the last imaging session and GDNF, Clover, and BFP production were evaluated by fluorescent immunohistochemistry (Figure 4). Transplants in animals terminated on dox (Figure 4A) and littermates terminated on dox (Figure 4B) significantly increased BFP expression in the striatal region. + Cells were shown to be upregulated in the dox-treated animals, whereas Clover and GDNF were only detected in the dox-treated animals. High amplification indicated that host cells adjacent to the transplant site could take up GDNF in the dox-treated animals (Figure 4C, pink arrow), whereas this was not observed in the off-dox animals (Figure 4D). Collectively, these data demonstrated that GDNF secretion can be induced and reversed in iNPCs several months after transplantation.

[0062] Finally, the level and location of GDNF expression was determined using DAB staining, which provides high sensitivity for protein detection and effectively tracks human GDNF-expressing NPC transplants (Figure 6 shows DAB staining of littermate animals from Figure 4). Blinded analysis revealed that all animals completed on-dox showed increased GDNF signal, except for one (animal no. 8), and none of the animals completed off-dox (Table 2).

[0063] Table 2. Blinded post-mortem GDNF scoring TIFF2024073654000003.tif149156(-) no positive staining, (+) slight staining, (++) moderate staining, (+++) widespread staining. Str=striatum. CC=corpus callosum.

[0064] This analysis also revealed that variations in bioluminescence intensity could be a result of changes in the location of the graft. Specifically, some animals that showed significantly higher bioluminescence signals had transplanted cells in the meninges, which could be due to backflow of cells into the injection tract during needle retraction (Figure 7). Overall, extensive analysis of all study animals confirmed a strong correlation between bioluminescence signal and GDNF expression.

[0065] Example 12 Consideration This report provides proof of concept that GDNF produced by engineering human neural progenitor cells can be tightly controlled over multiple cycles in vivo. This technology can also be applied to other growth factors, providing a valuable means of protecting neurons damaged in various diseases. Using pB-RTP-Tet-GDNF / memClover-FLuc, we observed that dox penetrated the substantial implantation site to levels that allowed transgene activation from the inducible TRE-Bi promoter. We also report that transgene activity can be induced and reverted over multiple cycles. Thus, if patients develop side effects, desensitization, or other transgene-related phenomena during disease progression, GDNF administration can be attenuated or stopped. Alternatively, drug regimens can be altered to allow resensitization of host receptors and signaling pathways. This is in stark contrast to ex vivo gene therapy, which involves constitutive gene expression. Importantly, the amount of dox concentration required for gene regulation (15 μg dox / g weight) is less than the antibacterial dose and does not increase the presence of dox-resistant bacteria or adversely affect the gut microbiota.

[0066] Interestingly, compared to lentiviral transduction of human NPCs used in current ALS clinical trials, the novel dox-catalyzed system provides nearly two-fold higher GDNF secretion. This increase may be due to piggyBac transposition, which may mediate more copies of the plasmid genome insert than lentiviral transduction. Furthermore, GDNF transcription in previous lentiviral experiments depends on the PGK promoter, which is activated by endogenous transcription factors. In this study, GDNF transcription is initiated by the binding of the Tet-On transactivator to the TRE-Bi promoter, which is catalyzed by a conformational change initiated by dox administration. Thus, transcription is not limited by naturally occurring transcription factors, but rather by the amount of transactivators and dox present. It is important to note that transplantation of ex vivo engineered neural progenitor cells, as performed in this study, increases the level of safety compared to direct genetic manipulation of host cells by viral transduction of pB-RTP-Tet-GDNF / memClover-Flu. Specifically, direct viral transduction into the brain can not only target healthy cells adjacent to diseased cells, but also impair diseased host cells. Moreover, by itself, the simple introduction of healthy neural progenitor / stem cells can have beneficial effects on the diseased host environment of various diseases. Thus, the synergistic effect of both cell therapy and inducible gene therapy can exceed gene therapy alone. Especially when the neural cells are derived from a human iPSC source, it offers the definitive promise of autologous transplantation.

[0067] Based on brain examination, none of the animals showed abnormal behavior or any overt damage to the transplanted striatum. Importantly, no tumors or ectopic tumors were observed in any of the transplanted animals in this study or in our previous iPSC transplantation studies. However, further safety and efficacy testing will be required to transfer this therapy to the clinic. First, the long-term efficacy, toxicity, and potential antigenicity of the constitutively expressed transactivator (rtTA-V10) must be tested. Second, we used pBase in our study as a means to stably integrate pB-RTP-Tet-GDNF / memClover-FLuc because of its ease of use. Although pBase mediates random integration, we have not seen tumors in over 500 mice in which pBase was used. However, a clinical grade product may require site-specific integration of a single copy. To this end, targeting AAVS1 safe landing may enable long-term stable integration and methylation-resistant expression of the transgene. Importantly, we observe significant dox-mediated transgene expression at the single copy level using the same TRE-Bi promoter used in this study.

[0068] In conclusion, this proof-of-principle study lays the foundation for an inducible combination of gene and cell therapy that functions to provide protection for the treatment of neurodegenerative diseases.

[0069] Example 13 Inducible expression of human GDNF from the AAVS1 "safe landing site" in human iPSC cell lines Application of the above constructs in a therapeutic setting would greatly benefit from "safe" integration to avoid adverse effects. This would include the development of a DNA vector producing human GDNF, optionally with regulatable expression, that could be targeted for integration into the human AAVS1 locus.

[0070] As a starting point, we use the human GDNF production plasmid pB-RTP-Tet-GDNF / memClover-FLuc from Breunig Laboratory, CSMC, mentioned above. The AAVS1 SA-2A-puro-pA donor plasmid was a gift from Rudolf Jaenisch (Addgene plasmid number 22075, http: / / n2t.net / addgene:22075, RRID:Addgene_22075). To facilitate the cloning of pB-RTP-Tet-GDNF / memClover-FLuc between the AAVS1 homologous sequences, the AAVS1 SA-2A-puro-pA donor plasmid can be modified to include an SfiI restriction site. This new intermediate vector is called AAVS1-AVPT.

[0071] The intermediate vectors AAVS1-AVPT and pB-RTP-Tet-GDNF / memClover-FLuc were digested with restriction enzymes SalI and SfiI. The appropriate fragments were isolated and joined by DNA ligation. The resulting construct was confirmed by DNA sequencing and tentatively named AAVS1-teton-hGDNF. As shown in FIG. 8 [SEQ ID NO: 2]. The AAVS1-teton-hGDNF construct was introduced into human iPSC line CS0002iCTR-n11 by electroporation. Targeted integration into the AAVS1 locus is achieved by including plasmids hAAVS1 1R TALEN and hAAVS1 1L TALEN (hAAVS1 1R TALEN was a gift from Feng Zhang (Addgene plasmid no. 35432, http: / / n2t.net / addgene:35432, RRID:Addgene_35432) hAAVS1 1L TALEN was a gift from Feng Zhang (Addgene plasmid no. 35431, http: / / n2t.net / addgene:35431, RRID:Addgene_35431)).

[0072] Example 14 Expression of GDNF in iPSC cells To facilitate identification of AAVS1-teton-hGDNF-containing cells based on expression of the fluorescent protein Clover, cultures are maintained in the presence of doxycycline, a tetracycline analog. "Green" colonies were picked under a fluorescent microscope for growth. Once sufficient numbers of cells were generated, clones were further purified by FACS sorting. At the same time, media was collected from these clones to confirm GDNF production by ELISA assay.

[0073] Example 15 Clones of single copy variants of GDNF expression plasmids (TREBi and TRB3 inducible promoters) Another example of the above includes single copy variants of GDNF expression plasmids. Using inducible promoters in TREBi and TRB3, such vectors can include reports such as eGFP and Luc2. Examples include pDonor-Teton3g-2a-TagBFP-V5-nls-p2a-puroR WPRE_isolated mpclover-2a-luc2pest-2a-gdnf wpre [SEQ ID NO: 3] shown in Figure 10. Such vectors include homologous recombination sequences. Here, AAVS1 targeting of the endogenous locus is between exon 1 and exon 2 of the human PPP1R12C gene. The recipient "landing site" contained a reporter / selection cassette (TagBFP2 and PuroR for fluorescence and antibiotic selection) driven by a splice acceptor linked to the upstream PPP1R12C, and a constitutive CAG promoter-driven td-Tomato red fluorescent cistron flanked by LoxP and FRT sites was stably integrated. The cells were then lipofected with plasmids expressing FlpO and Cre upon stable selection for these reporters, and the donor plasmid contained the LoxP- and FRT-flanked selection / reporter cassette, as well as a dox-inducible mpClover / Luc2p / GDNF cistron-containing plasmid.

[0074] Transmitted light and green fluorescent imaging of cells in the absence and presence of doxycycline shows inducibility of mpClover 24 hours after dox addition, and the absence of GFP "leakage" in the absence of dox. It should be noted that this population is not a pure population, as it was not puroselected (thus negative cells do not contain donor factors). Additionally, the cells were tested approximately one week after sorting to enrich for transfected cells, and two weeks after transfection, to ensure that the mpClover population is a stable transfectant. Dox inducibility would not function in the absence of integration.

[0075] The above various methods and techniques provide multiple ways of implementing the present invention. Of course, it should be understood that not all of the stated objectives or advantages are necessarily achieved according to any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the method can be implemented in a manner that achieves or optimizes one advantage or advantages of the teachings herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. Various advantageous and disadvantageous alternatives are described herein. It should be understood that some of the preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically alleviate the disadvantageous features by including one, another, or several advantageous features.

[0076] Moreover, one skilled in the art will recognize the applicability of various features in different embodiments. Similarly, the various elements, features, and steps described above, as well as other known equivalents for each such element, feature, or step, can be combined by one skilled in the art to perform a method in accordance with the principles described herein. In various embodiments, the various elements, features, and steps will include some specifically and exclude others specifically.

[0077] Although the present invention is disclosed in the context of certain embodiments and examples, it will be appreciated by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, as well as modifications and equivalents thereof.

[0078] Many variations and alternative elements are disclosed in the embodiments of the present invention. Further variations and alternative elements will be apparent to those skilled in the art. These variations include, but are not limited to, compositions and methods related to induced pluripotent stem cells (iPSCs), differentiated iPSCs, including neural progenitor cells, vectors used to manipulate the above-mentioned cells, methods and compositions related to the use of the above-mentioned compositions, methods and compositions and solutions used therein, and specific uses of products made according to the teachings of the present invention. Various embodiments of the present invention may specifically include or exclude any of these variations or elements.

[0079] In some embodiments, the numbers expressing properties such as amounts of ingredients, concentrations, reaction conditions, and the like, used to describe and claim certain embodiments of the present invention should be understood to be modified in some cases by the term "about". Thus, in some embodiments, the numerical parameters set forth in the written description and accompanying claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. The numerical values ​​presented in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0080] In some embodiments, the terms "a" and "an" and "the" and similar referents used in the context of describing certain embodiments of the invention (especially in the specific context of the claims below) can be interpreted to cover both the singular and the plural. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein with respect to specific embodiments is intended to better illustrate the invention and does not impose limitations on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0081] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group so modified herein and satisfies the written description of all Markush groups used in the appended claims.

[0082] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the above description. It is contemplated that those skilled in the art may employ such variations, if desired, and the invention may be practiced other than as specifically described herein. Accordingly, many embodiments of the invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0083] Additionally, throughout this specification, numerous references are made to patents and printed publications. Each of the above cited references and printed publications is individually incorporated herein by reference in its entirety.

[0084] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the embodiments of the invention are not limited to exactly as shown and described.

[0085] Sequence information SEQUENCE LISTING <110> CEDARS-SINAI MEDICAL CENTER <120> METHODS AND COMPOSITIONS FOR INDUCIBLE EXPRESSION OF NEUROTROPHIC FACTORS <150> US 62 / 644,332 <151> 2018-03-16 <150> US 62 / 773,752 <151> 2018-11-30 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 12962 <212> DNA <213> Artificial Sequence <220> <223> pB-RTP-Tet-GDNF / memClover-FLuc <400> 1 gtcgacttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta 60 aaattgacgc atgtgtttta tcgatctgta tatcgaggtt tattatta tttgaataga 120 tattagttt tattatattt acacttacat actaata aattchcaca acaatttatt 180 tatgtttatt tattatta aaaaaaaaaaactcaaa tttctctat aaagtaaca 240 aacttttact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 300 ggagttccgc gttacataac ttacggtaa tggcccgcct ggctgaccgc ccaacgaccc 360 ccgcccattg acgtcaataa tgacgtagt tcccatagta acgccaatag ggactttcca 420 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 480 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 540 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 600 cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc 660 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatgggg 720 cggggggggggggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg 780 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tcctttatg 840 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct 900 gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc ccggctctga 960 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 1020 tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag ccttaaaggg 1080 ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg 1140 cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg cgggcgcggc 1200 gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccggggc ggtgccccgc 1260 ggtgcgggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg 1320 agcaggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc cctccccgag 1380 ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc gcggggctcg 1440 ccgtgccggg cgggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg 1500 ccggggaggg ctcggggag gggcgcggcg gccccccggag cgccggcggc tgtcgaggcg 1560 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt 1620 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcacccc tctagcgggc 1680 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 1740 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct 1800 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta 1860 gagcctctgc taaccatgtt catgccttct tcttttcct acagctcctg ggcaacgtgc 1920 tggttattgt gctgtctcat cattttggca aagaattcgg taccgcatgc atcgatgcta 1980 gcgcgccgcc accatgtcta gactggacaa gagcaaagtc ataaactctg ctctggaatt 2040 actcaatgga gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt 2100 tgagcagcct accctgtact ggcacgtgaa gaaagcgg gccctgctcg atgccctgcc 2160 aatcgagatg ctggacaggc atcataccca ctcctgcccc ctggaaggcg agtcatggca 2220 agactttctg cggaacg ccaagtcata ccgctgtgct ctcctctcac atcgcgacgg 2280 ggctaaagtg catctcggca cccgcccaac agaaacag tacgaaaccc tggaaaatca 2340 gctcgcgttc ctgtgtcagc aaggctttc cctggagaac gcactgtacg ctctgtccgc 2400 cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga 2460 ggaagagag acacctacca ccgattctat gccccccactt ctgaaacaag caattgagct 2520 gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg 2580 cctggagaaa cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg acgattttga 2640 cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga 2700 cgctcttgac gattttgacc ttgacatgct ccccggggga tccggaggat ccggagccac 2760 gaacttctct ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ctatgagcga 2820 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca 2880 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat 2940 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct 3000 ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc 3060 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac 3120 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg 3180 ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt 3240 caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa 3300 gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc acatatagat ccaagaaacc 3360 cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa 3420 ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga 3480 cctccctagc aaactggggc acagcttta ttccggactc ggcaagccta tccctaaccc 3540 tctgctgggc ctggacagca ccgatccaa aaagagaga aaggtagacc ctaagaagaa 3600 gaggaaggtg gacccaga agagagaaa ggtgtgaggc agtgaggga gtggagcaac 3660 caatttttca ctcctgaagc aagctgggga tgtagaggag aatcctgggc ctatggacta 3720 haaagacgat gacgacaagc tggcactag tggcttgcg atgaattgg gacctaggtt 3780 gatgggcaag ctttctagtc aactcgagat gaccgagtac aagcccacgg tgcgcctcgc 3840 cacccgcgac gacgccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc 3900 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga 3960 actctccctc acgcgcgtcg ggctcgacat cggcaggtg tggtcgcgg acgacggcgc 4020 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat 4080 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcac agatggaagg 4140 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc 4200 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc 4260 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta 4320 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg 4380 gtgcatgacc cgcaagcccg gtgcctgagc ggccgcactc ctcaggtgca ggctgcctat 4440 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt 4500 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa 4560 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga 4620 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg 4680 gcaacacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag 4740 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt 4800 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc 4860 ttacatgttt tactagccag attttcctc ctctcctgac tactcccagt catagctgtc 4920 cctcttctct tatggagatc cctcgacctg ggtaacgcca gggttttcc agtcacgacg 4980 ttgtaaaacg acggccagtg ccaagcttgc atgcctgcag taagatacat tgatgagttt 5040 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 5100 attgctttat ttgtaacat tataagctgc aataaacaag ttaacaacaa caattgcatt 5160 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc 5220 tacaaatgtg gtaggtacca ccggtcctgc aggttagacg ttgatcctgg cgctggcgca 5280 agcagcaggg tgtctatcca tgccgctctc ctgggcgcag ctcatgggca gggtgccggc 5340 ggcctgctcc tccacctcgg gagggaagcc gtgagaattc acggcgatct tgccgccctt 5400 cttggcctta atgagaatct cgcggatctt gcgggcgtcc aacttgccgg tcagtccttt 5460 aggcacctcg tccacgaaca caacaccacc gcgcagcttc ttggcggttg taacctggct 5520 ggccacatag tccacgatct ccttctcggt catggtttta ccgtgttcca gcacgacgac 5580 tgcggcgggc agctcgccgg catcgtcgtc gggcaggccg gcgaccccgg cgtcgaagat 5640 gttggggtgt tgcagcagga tgctctccag ttcggctggg gctacctggt agcccttgta 5700 tttgatcagg ctcttcagcc ggtccacgat gaagaagtgc tcgtcctcgt cccagtaggc 5760 gatgtcgccg ctgtgcagcc agccgtcctt gtcgatgaga gcgtttgtag cctcggggtt 5820 gttaacgtag ccgctcatga tcatggggcc acggacgcac agctcgccgc gctggttcac 5880 acccagtgtc ttaccggtgt ccaagtccac caccttagcc tcgaagaagg gcaccacctt 5940 gcctactgcg ccaggcttgt cgtccccttc gggggtgatc agaatggcgc tggttgtttc 6000 tgtcaggccg tagccctggc ggatgcctgg taggtggaag cgtttggcca cggcctcacc 6060 tacctccttg ctgagcggcg ccccgccgct ggcgatctcg tgcaagttgc ttaggtcgta 6120 cttgtcgatg agagtgctct tagcgaagaa gctaaatagt gtgggcacca gcagggcaga 6180 ttgaatctta tagtcttgca agctgcgcaa gaatagctcc tcctcgaagc ggtacatgag 6240 6300 aaatggcacc acgctgagga tagcggtgtc ggggatgatc tggttgccga agatggggtc 6360 gcgggcatga ctgaatcgga cacaagcggt gcggtgcggt agggctacgc ccttgggcaa 6420 tccggtactg ccactactgt tcatgatcag ggcgatggtt ttgtcccggt cgaagctctc 6480 gggcacgaag tcgtactcgt tgaagccggg tggcaaatgg gaagtcacga aggtgtacat 6540 gctttggaag ccctggtagt cggtcttgct atccatgatg atgatctttt gtatgatcgg 6600 tagcttcttt tgcacgttga ggatcttttg cagccctttc ttgctcacga atacgacggt 6660 gggctggctg atgcccatgc tgttcagcag ctcgcgctcg ttgtagatgt cgttagctgg 6720 ggccacagcc acaccgatga acagggcacc caacacgggc atgaagaact gcaagctatt 6780 ctcgctgcac accacgatcc gatggtttgt attcagccca tagcgcttca tagcttctgc 6840 cagccgaacg ctcatctcga agtactcggc gtaggtaatg tccacctcga tatgtgcgtc 6900 ggtaaaggcg atggtgccgg gcaccagggc gtagcgcttc atggctttgt gcagctgctc 6960 gccggcggtc ccgtcttcga gtgggtagaa tggcgctggg cccttcttaa tgtttttggc 7020 atcttccata ggaccggggt tttcttccac gtctcctgct tgctttaaca gagagaagtt 7080 cgtggctccg gatccggcgg cggttacgaa ctccagcagg accatgtgat cgcgcttctc 7140 gttggggtct ttgctcaggg cggactgatg gctcaggtag tggttgtcgg gcagcagcac 7200 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc 7260 ctcaacgttg tggcggatct tgaagttagc cttgatgccg ttcttctgct tgtcggccgt 7320 gatatagacg ttgtggctgt tgaagttgta ctccagcttg tgccccagga tgttgccgtc 7380 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt 7440 cacctcggcg cgggtcttgt aggtaccgtc gtccttgaaa gagatggtgc gctcctggac 7500 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg 7560 gctgaagcag gccacgccgt agccgaaggt ggtcacgagg gtgggccagg gcacgggcag 7620 cttgccggtg gtgcagatga acttcagggt cagcttgccg ttggtggcat cgccctcgcc 7680 ctcgccgcgg acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg 7740 caccaccccg gtgaacagct cctcgccctt gctcaccatg tccacgccgt cgtcgttcag 7800 gttgtccttg cgcttgctct tgatgcagcc catggtggcg gcggtcacca cgcgtccgcg 7860 atctgacggt tcactaaacg agctctgctt atataggcct cccaccgtac acgccacctc 7920 gacatactcg agtttactcc ctatcagtga tagagaacgt atgaagagtt tactccctat 7980 cagtgataga gaacgtatgc agactttact ccctatcagt gatagaagaac gtataaggag 8040 tttactccct atcagtgata gagaacgtat gaccagttta ctccctatca gtgatagaga 8100 acgtatctac agtttactcc ctatcagtga tagagaacgt atatccagtt tactccctat 8160 cagtgataga gaacgtataa gctttaggcg tgtacggtgg gcgcctataa aagcagagct 8220 cgtttagtga accgtcagat cgcctggagc aattccacaa cacttttgtc ttataccaac 8280 tttccgtacc acttcctacc ctcgtaaaca gctggccgcc accatgaagt tatgggatgt 8340 cgtggctgtc tgcctggtgc tgctccacac cgcgtccgcc ttcccgctgc ccgccggtaa 8400 gaggcctccc gaggcgcccg ccgaagaccg ctccctcggc cgccgccgcg cgcccttgcg 8460 gctgagcagt gactcaaata tgccagagga ttatcctgat cagttcgatg atgtcatgga 8520 ttttattcaa gccaccatta aaagactgaa aaggtcacca gataaacaaa tggcagtgct 8580 tcctagaaga gagcggaatc ggcaggctgc agctgccaac ccagagaatt ccagaggaaa 8640 aggtcggaga ggccagaggg gcaaaaaccg gggttgtgtc ttaactgcaa tacatttaaa 8700 tgtcactgac ttgggtctgg gctatgaaac caaggaggaa ctgattttta ggtactgcag 8760 cggctcttgc gatgcagctg agacaacgta cgacaaaata ttgaaaaact tatccagaaa 8820 tagaaggctg gtgagtgaca aagtagggca ggcatgttgc agacccatcg cctttgatga 8880 tgacctgtcg tttttagatg ataacctggt ttaccatatt ctaagaaagc attccgctaa 8940 aaggtgtgga tgtatctgat tcgaaagccg cactcctcat aatcaacctc tggattacaa 9000 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 9060 cgctgcttta atgcctttgt atcatgctat tgcttccccgt atggctttca tttctctctc 9120 cttgtataaa tcctggttgc tgtctcttta tgaggatttg tggcccgttg tcaggcaacg 9180 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 9240 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 9300 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 9360 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 9420 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 9480 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 9540 tcggatctcc ctttgggccg cctccccgca ggccgcacac ctcaggtgca ggctcgaaat 9600 ttaaatcctc gagggccgca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg 9660 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta 9720 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt 9780 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc 9840 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgccatatg 9900 ctggctgcca tgacaagg tggctataaa gaggtcatca gtatatgaa cagccccctg 9960 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagatttt ttatatttt 10020 gttttgtgtt atttttttct ttacatccc taaaatttc cttacatgtt ttactagcca 10080 gatttttcct cctctcctga ctactcccag tcatagctgt ccctctctc ctgcagatat 10140 ctataacaag aaatatata tataaagt tatcacgtaa gtagaacatg aaatacaat 10200 ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gatacatg cgtcattttg 10260 actcacgcgg tcgttagt tcaaatcag tgacacttac cgcattgaca agcacgccctc 10320 acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt 10380 tagaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct 10440 agggttaagc ttggcgtaat catggtcata gctgttttcct gtgtgaaatt gttatccgct 10500 cacaatcca cacacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 10560 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gcttccagt cgggaaacct 10620 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 10680 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 10740 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 10800 ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata 10860 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 10920 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca 10980 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 11040 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 11100 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 11160 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 11220 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 11280 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 11340 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 11400 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 11460 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 11520 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 11580 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 11640 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 11700 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 11760 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 11820 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 11880 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 11940 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 12000 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 12060 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 12120 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 12180 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 12240 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 12300 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 12360 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 12420 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 12480 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 12540 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 12600 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 12660 actctcagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcaccca 12720 ctgatcttca gcatcttta ctttcaccag cgtttctggg tgagcaaaa caggaaggca 12780 aaatgccgca aaaaagggaa taagggcgac acggaatgt tgaatactca tactctctcct 12840 tttcaat tattgagca tttcaggg ttattgtctc atgagcggat acatatttga 12900 atgtatttag aaaaataac aaataggggt tccgcgcaca ttcccgaa aagtgccacc 12960 tg 12962 <210> 2 <211> 16438 <212> DNA <213> Artificial Sequence <220> <223> AAVS1-teton-Hgdnf <400> 2 gtcgacttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta 60 aaattgacgc atgtgtttta tcgatctgta tatcgaggtt tattatta tttgaataga 120 tattagttt tattatattt acacttacat actaata aattchcaca acaatttatt 180 tatgtttatt tattatta aaaaaaaaaaactcaaa tttctctat aaagtaaca 240 aacttttact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 300 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 360 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 420 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 480 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 540 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 600 cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc 660 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatgggg 720 cggggggggggggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg 780 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tcctttatg 840 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct 900 gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc ccggctctga 960 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 1020 tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag ccttaaaggg 1080 ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg 1140 cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg cgggcgcggc 1200 gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc 1260 ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg 1320 agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc cctccccgag 1380 ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc gcggggctcg 1440 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg 1500 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg 1560 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt 1620 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc 1680 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 1740 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct 1800 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta 1860 gagcctctgc taaccatgtt catgccttct tcttttcct acagctcctg ggcaacgtgc 1920 tggttattgt gctgtctcat cattttggca aagaattcgg taccgcatgc atcgatgcta 1980 gcgcgccgcc accatgtcta gactggacaa gagcaaagtc ataaactctg ctctggaatt 2040 actcaatgga gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt 2100 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc 2160 aatcgagatg ctggacaggc atcataccca ctcctgcccc ctggaaggcg agtcatggca 2220 agactttctg cggaacaacg ccaagtcata ccgctgtgct ctcctctcac atcgcgacgg 2280 ggctaaagtg catctcggca cccgcccaac agaagaaacag tacgaaaccc tggaaaatca 2340 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc 2400 cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga 2460 ggaaagagag acacctacca ccgattctat gcccccactt ctgaaacaag caattgagct 2520 gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg 2580 cctggagaaa cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg acgattttga 2640 cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga 2700 cgctcttgac gattttgacc ttgacatgct ccccggggga tccggaggat ccggagccac 2760 gaacttctct ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ctatgagcga 2820 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca 2880 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat 2940 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct 3000 ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc 3060 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac 3120 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg 3180 ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt 3240 caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa 3300 gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc acatatagat ccaagaaacc 3360 cgctaagaac ctcaagatgc ctggcgcta ctatgtggac tacagactgg aaagaatcaa 3420 ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga 3480 cctccctagc aaactggggc acaagcttaa ttccggactc ggcaagccta tccctaaccc 3540 tctgctgggc ctggacagca ccgatccaaa aaagaagaga aaggtagacc ctaagaagaa 3600 gaggaaggtg gaccccaaga agaagagaaaa ggtgtgaggc agtggaggga gtggagcaac 3660 caatttttca ctcctgaagc aagctgggga tgtagaggag aatcctgggc ctatggacta 3720 caaagacgat gacgacaagc ttggcactag tggctttgcg aatgaattgg gacctaggtt 3780 gatgggcaag ctttctagtc aactcgagat gaccgagtac aagcccacgg tgcgcctcgc 3840 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc 3900 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga 3960 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc 4020 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat 4080 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg 4140 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc 4200 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc 4260 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta 4320 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg 4380 gtgcatgacc cgcaagcccg gtgcctgagc ggccgcactc ctcaggtgca ggctgcctat 4440 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt 4500 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa 4560 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga 4620 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg 4680 gcaacacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag 4740 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt 4800 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc 4860 ttacatgttt tactagccag attttcctc ctctcctgac tactcccagt catagctgtc 4920 cctcttctct tatggagatc cctcgacctg ggtaacgcca gggttttcc agtcacgacg 4980 ttgtaaaacg acggccagtg ccaagcttgc atgcctgcag taagatacat tgatgagttt 5040 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 5100 attgctttat ttgtaacat tataagctgc aataaacaag ttaacaacaa caattgcatt 5160 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc 5220 tacaaatgtg gtaggtacca ccggtcctgc aggttagacg ttgatcctgg cgctggcgca 5280 agcagcaggg tgtctatcca tgccgctctc ctgggcgcag ctcatgggca gggtgccggc 5340 ggcctgctcc tccacctcgg gagggaagcc gtgagaattc acggcgatct tgccgccctt 5400 cttggcctta atgagaatct cgcggatctt gcgggcgtcc aacttgccgg tcagtccttt 5460 aggcacctcg tccacgaaca caacaccacc gcgcagcttc ttggcggttg taacctggct 5520 ggccacatag tccacgatct ccttctcggt catggtttta ccgtgttcca gcacgacgac 5580 tgcggcgggc agctcgccgg catcgtcgtc gggcaggccg gcgaccccgg cgtcgaagat 5640 gttggggtgt tgcagcagga tgctctccag ttcggctggg gctacctggt agcccttgta 5700 tttgatcagg ctcttcagcc ggtccacgat gaagaagtgc tcgtcctcgt cccagtaggc 5760 gatgtcgccg ctgtgcagcc agccgtcctt gtcgatgaga gcgtttgtag cctcggggtt 5820 gttaacgtag ccgctcatga tcatggggcc acggacgcac agctcgccgc gctggttcac 5880 acccagtgtc ttaccggtgt ccaagtccac caccttagcc tcgaagaagg gcaccacctt 5940 gcctactgcg ccaggcttgt cgtccccttc gggggtgatc agaatggcgc tggttgtttc 6000 tgtcaggccg tagccctggc ggatgcctgg taggtggaag cgtttggcca cggcctcacc 6060 tacctccttg ctgagcggcg ccccgccgct ggcgatctcg tgcaagttgc ttaggtcgta 6120 cttgtcgatg agagtgctct tagcgaagaa gctaaatagt gtgggcacca gcagggcaga 6180 ttgaatctta tagtcttgca agctgcgcaa gaatagctcc tcctcgaagc ggtacatgag 6240 cacgacccga aagccgcaga tcaagtagcc cagcgtggtg aacatgccga agccgtggtg 6300 aaatggcacc acgctgagga tagcggtgtc ggggatgatc tggttgccga agatggggtc 6360 gcgggcatga ctgaatcgga cacaagcggt gcggtgcggt agggctacgc ccttgggcaa 6420 tccggtactg ccactactgt tcatgatcag ggcgatggtt ttgtcccggt cgaagctctc 6480 gggcacgaag tcgtactcgt tgaagccggg tggcaaatgg gaagtcacga aggtgtacat 6540 gctttggaag ccctggtagt cggtcttgct atccatgatg atgatctttt gtatgatcgg 6600 tagcttcttt tgcacgttga ggatcttttg cagccctttc ttgctcacga atacgacggt 6660 gggctggctg atgcccatgc tgttcagcag ctcgcgctcg ttgtagatgt cgttagctgg 6720 ggccacagcc acaccgatga acagggcacc caacacgggc atgaagaact gcaagctatt 6780 ctcgctgcac accacgatcc gatggtttgt attcagccca tagcgcttca tagcttctgc 6840 cagccgaacg ctcatctcga agtactcggc gtaggtaatg tccacctcga tatgtgcgtc 6900 ggtaaaggcg atggtgccgg gcaccagggc gtagcgcttc atggctttgt gcagctgctc 6960 gccggcggtc ccgtcttcga gtgggtagaa tggcgctggg cccttcttaa tgtttttggc 7020 atcttccata ggaccggggt tttcttccac gtctcctgct tgctttaaca gagagaagtt 7080 cgtggctccg gatccggcgg cggttacgaa ctccagcagg accatgtgat cgcgcttctc 7140 gttggggtct ttgctcaggg cggactgatg gctcaggtag tggttgtcgg gcagcagcac 7200 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc 7260 ctcaacgttg tggcggatct tgaagttagc cttgatgccg ttcttctgct tgtcggccgt 7320 gatatagacg ttgtggctgt tgaagttgta ctccagcttg tgccccagga tgttgccgtc 7380 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt 7440 cacctcggcg cgggtcttgt aggtaccgtc gtccttgaaa gagatggtgc gctcctggac 7500 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg 7560 gctgaagcag gccacgccgt agccgaaggt ggtcacgagg gtgggccagg gcacgggcag 7620 cttgccggtg gtgcagatga acttcagggt cagcttgccg ttggtggcat cgccctcgcc 7680 ctcgccgcgg acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg 7740 caccaccccg gtgaacagct cctcgccctt gctcaccatg tccacgccgt cgtcgttcag 7800 gttgtccttg cgcttgctct tgatgcagcc catggtggcg gcggtcacca cgcgtccgcg 7860 atctgacggt tcactaaacg agctctgctt atataggcct cccaccgtac acgccacctc 7920 gacatactcg agtttactcc ctatcagtga tagagaacgt atgaagagtt tactccctat 7980 cagtgataga gaacgtatgc agactttact ccctatcagt gatagaagaac gtataaggag 8040 tttactccct atcagtgata gagaacgtat gaccagttta ctccctatca gtgatagaga 8100 acgtatctac agtttactcc ctatcagtga tagagaacgt atatccagtt tactccctat 8160 cagtgataga gaacgtataa gctttaggcg tgtacggtgg gcgcctataa aagcagagct 8220 cgtttagtga accgtcagat cgcctggagc aattccacaa cacttttgtc ttataccaac 8280 tttccgtacc acttcctacc ctcgtaaaca gctggccgcc accatgaagt tatgggatgt 8340 cgtggctgtc tgcctggtgc tgctccacac cgcgtccgcc ttcccgctgc ccgccggtaa 8400 gaggcctccc gaggcgcccg ccgaagaccg ctccctcggc cgccgccgcg cgcccttgcg 8460 gctgagcagt gactcaaata tgccagagga ttatcctgat cagttcgatg atgtcatgga 8520 ttttattcaa gccaccatta aaagactgaa aaggtcacca gataaacaaa tggcagtgct 8580 tcctagaaga gagcggaatc ggcaggctgc agctgccaac ccagagaatt ccagaggaaa 8640 aggtcggaga ggccagaggg gcaaaaaccg gggttgtgtc ttaactgcaa tacatttaaa 8700 tgtcactgac ttgggtctgg gctatgaaac caaggaggaa ctgattttta ggtactgcag 8760 cggctcttgc gatgcagctg agacaacgta cgacaaaata ttgaaaaact tatccagaaa 8820 tagaaggctg gtgagtgaca aagtagggca ggcatgttgc agacccatcg cctttgatga 8880 tgacctgtcg tttttagatg ataacctggt ttaccatatt ctaagaaagc attccgctaa 8940 aaggtgtgga tgtatctgat tcgaaagccg cactcctcat aatcaacctc tggattacaa 9000 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 9060 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 9120 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 9180 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 9240 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 9300 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 9360 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 9420 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 9480 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 9540 tcggatctcc ctttgggccg cctccccgca ggccgcacac ctcaggtgca ggctcgaaat 9600 ttaatcctc gagggccgca ctcctcaggt gcaggctgcc tatcagagg tggtggctgg 9660 tgtggccaat gccctggctc acaaatacca ctgagatctt ttccctg ccaaaaatta 9720 tggggacatc atgaagcccc tggcatct gactctctggc tataaagga aatttattt 9780 cattgcaata gtgtgttgga atttttgtg tctctcactc ggaaggacat atgggaggc 9840 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcacat atgccatatg 9900 ctggctgcca tgacaagg tggctataaa gaggtcatca gtatatgaa cagccccctg 9960 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagatttt ttatatttt 10020 gttttgtgtt atttttttct ttacatccc taaaatttc cttacatgtt ttactagcca 10080 gatttttcct cctctcctga ctactcccag tcatagctgt ccctctctc ctgcagatat 10140 ctataacaag aaatatata tataaagt tatcacgtaa gtagaacatg aaatacaat 10200 ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gatacatg cgtcattttg 10260 actcacgcgg tcgttagt tcaaatcag tgacacttac cgcattgaca agcacgccctc 10320 acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt 10380 tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct 10440 agggttaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 10500 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 10560 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 10620 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 10680 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 10740 ttttttattt atgcagaggc cgaggccgcc tcggccagat ctctcgaggc cctgtgggag 10800 gaagagaaga ggtcagaagc ttgccccact gtggggtgga ggggacagat aaaagtaccc 10860 agaaccagag ccacattaac cggccctggg aatataaggt ggtcccagct cggggacaca 10920 ggatccctgg aggcagcaaa catgctgtcc tgaagtggac ataggggccc gggttggagg 10980 aagaagacta gctgagctct cggacccctg gaagatgcca tgacaggggg ctggaagagc 11040 tagcacagac tagagaggta aggggggtag gggagctgcc caaatgaag gagtgagagg 11100 tgacccgaat ccacaggaga acggggtgtc caggcaaaga aagcaagagg atggagaggt 11160 ggctaaagcc agggagacgg ggtactttgg ggttgtccag aaaaacggtg atgatgcagg 11220 cctacaagaa ggggaggcgg gacgcaaggg agacatccgt cggagaagc catcctaaga 11280 aacgagagat ggcacaggcc ccagaaggag aagaaaagg gaacccagcg agtgaagacg 11340 gcatggggtt gggtgaggga ggagagatgc ccggagag cccagacacg gggaggatcc 11400 11460 gggcagcaac acagcagaga gcaaggggaa gagggagtgg aggagaacgg aacctgaagg 11520 aggcggcagg gaaggatctg ggccagccgt agaggtgacc caggccacaa gctccagaca 11580 gaagcggca caggcccagg ggagagaatg ctggtcagag aaagcaaagg gcgaattcgt 11640 ttaaacctgc aggactagtc cctttagtga gggttaattc tgagcttggc gtaatcatgg 11700 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 11760 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 11820 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 11880 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 11940 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 12000 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 12060 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 12120 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 12180 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 12240 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 12300 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 12360 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 12420 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 12480 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 12540 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 12600 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 12660 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 12720 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagaca ataaccctga 12780 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 12840 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 12900 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 12960 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 13020 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 13080 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 13140 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 13200 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 13260 13320 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 13380 aaactattaa ctggcgaact acttactcta gcttccccggc aaaattaat agactggatg 13440 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 13500 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 13560 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 13620 gacgaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 13680 gaccaagttt actcatatat actttaagatt gatttaaaac ttcattttta atttaaaaagg 13740 atctaggtga agatcctttt tgataatctc atgagcggat acatatttga atgtatttag 13800 13860. aaaaataac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taggaaaa ataccgcatc aggaaattgt aagcgttaat aattcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 14160. aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg 14280. cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 14340 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 14460 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 14520 ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 14580 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 14640 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 14700 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag agcttgatcc 14760 cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc 14820 aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc 14880 ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct ttgcgcttgc 14940 gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg 15000 cggactggct ttctacgtga aaaggatcta ggtgaagatc ctttttgata atctcatgcc 15060 tgacatttat attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg 15120 gctgagatca gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt 15180 catcatgcgc cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt 15240 atctgacagc agacgtgcac tggccagggg gatcaccatc cgtcgccccg gcgtgtcaat 15300 aatatcactc tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac 15360 cttaaactgc cgtacgtata ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 15420 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac 15480 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc 15540 actatagggc gaattgaatt tagcggccgc gaattcgccc ttgcccttag agcagagcca 15600 ggaacccctg tagggaaggg gcaggagagc caggggcatg agatggtgga cgaggaaggg 15660 ggacagggaa gcctgagcgc ctctcctggg cttgccaagg actcaaaccc agaagcccag 15720 agcaggggcct tagggaagcg ggaccctgct ctgggcggag gaatatgtcc cagatagcac 15780 tggggactct ttaaggaaag aaggatggag aaagagaaag ggagtagagg cggccacgac 15840 ctggtgaaca cctaggacgc accattctca caagggagt tttccacacg gacacccccc 15900 tcctcaccac agccctgcca ggacggggct ggctactggc cttatctcac aggtaaaact 15960 gacgcacgga ggaacaatat aaattggga ctagaaaaggt gaagccaa agtttagaact 16020 caggaccaac ttatctgat tttgtttttc caaactgctt ctcctcttgg gaagtgtaag 16080 gaagctgcag caccaggatc agtgaaacgc accagacagc cgcgtcagag cagctcaggt 16140 tctgggagag ggtagcgcag ggtggccact gagaaccggg caggtcacgc atccccccct 16200 tccctcccac cccctgccaa gctctccctc ccaggatcct ctctggctcc atcgtaagca 16260 16320 taggaatct gcctaacagg aggtgggggt tagacccaat atcaggagac taggaaggag 16380 gaggcctaag gatggggctt ttctgtcacc aatcctgtcc ctagtaaagc ttagtact 16438 <210> 3 <211> 15067 <212> DNA <213> Artificial Sequence <220> <h2 style=";text-align:left;direction:ltr"><223> pDonor-Teton3g-2a-TagBFP-V5-nls-p2a-puroR WPRE_Insulated<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> mpclover-2a-luc2pest-2a-gdnf wpre<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggtcgacatt gattattgac tagtggatcc gctgtaagtc tgcagaaatt gatgatctat 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> taaacaataa agatgtccac taaaatggaa gtttttcctg tcatactttg ttaagaaggg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgagaacaga gtacctacat tttgaatgga aggattggag ctacgggggt gggggtgggg 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgggattaga taaatgcctg ctctttactg aaggctcttt actattgctt tatgataatg 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttcatagtt ggatatcata atttaaacaa gcaaaaccaa attaagggcc agctcattcc 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcccactcat gatctataga tctatagatc tctcgtggga tcattgtttt tctcttgatt 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cccactttgt ggttctaagt actgtggttt ccaaatgtgt cagtttcata gcctgaagaa 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cgagatcagc agcctctgtt ccacatacac ttcattctca gtattgtttt gccaagttct 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aattccatca gaagcttgca gatctgcgac tctagaggat ctgcgactct agaggatcat 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc 600<h2 style=";text-align:left;direction:ltr"> cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta 660 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 720 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgcga 780 ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac 840 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg 900 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 960 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 1020 gtctggatct gcgactctag aggatcataa tcagccatac cacatttgta gaggttttac 1080 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg 1140 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 1200 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 1260 atgtatctta tcatgtctgg atccccatca agctgatccg gaacccttaa tataacttcg 1320 tatagcatac attatacgaa gttattaggt ccctcgacct gcagcccaag ctagcttatc 1380 gatgctagcc agctgaccgg tggccgccac catgtctaga ctggacaaga gcaaagtcat 1440 aaactctgct ctggaattac tcaatggagt cggtatcgaa ggcctgacga caaggaaact 1500 cgctcaaaag ctgggagttg agcagcctac cctgtactgg cacgtaga acaagcgggc 1560 cctgctcgat gccctgccaa tcgagatgct ggacaggcat catacccact cctgccccct 1620 ggaaggcgag tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct 1680 cctctcacat cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta 1740 cgaaaccctg gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc 1800 actgtacgct ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga 1860 gcatcaagta gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct 1920 gaaaagca attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct 1980 ggaataatc atatgtggcc tggagaaa gctaaagtgc gaaagcggcg ggccgaccga 2040 cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg actttgacct 2100 tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccgggggatc 2160 cggaggatcc ggagccacga acttctctct gttaaagcaa gcaggagacg tggaagaaaa 2220 ccccggtcct atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg 2280 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg 2340 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat 2400 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc 2460 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga 2520 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta 2580 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac 2640 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag 2700 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaacg ccaagaccac 2760 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta 2820 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc 2880 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt ccggactcgg 2940 caagcctatc cctaaccctc tgctgggcct ggacagcacc gatccaaaaa agaagagaaa 3000 ggtagaccct aagaagaaga ggaaggtgga ccccaagaag aagagaaagg tgtgaggcag 3060 tggagggagt ggagcaacca atttttcact cctgaagcaa gctggggatg tagaggagaa 3120 tcctgggcct atggactaca aagacgatga cgacaagctt ggcactagtg gctttgcgaa 3180 tgaattggga cctaggttga tgggcaagct ttctagtcaa ctcgagatga ccgagtacaa 3240 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc 3300 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg 3360 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg 3420 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg 3480 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc 3540 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct 3600 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct 3660 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc 3720 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc 3780 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgatacg tagcggccgc 3840 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 3900 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 3960 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 4020 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 4080 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 4140 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 4200 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 4260 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 4320 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 4380 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc aggccgcact 4440 cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac 4500 aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt 4560 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat 4620 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat 4680 gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt 4740 tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat 4800 agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct 4860 ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga 4920 ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagatcctc 4980 tagcgataag cttgatatcg aattcgagtt ggcgcgcctg tcattctaaa tctctctttc 5040 agcctaaagc tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa 5100 gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct 5160 gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc 5220 tgccccctag cgggggaggg acgtaattac atccctgggg gctttgggag ggggctgtcc 5280 ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct ttcagcctaa agctttttcc 5340 ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg 5400 atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg 5460 gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga 5520 gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag ctcaataaaa 5580 gagcccacaa cccctcactc ggcgcgccag tcctccgata gactgcgtcg cccgggtacc 5640 cgtgtatcca ataaaccctc ttgcagttta tccagccctc actccttctc tagggcgatc 5700 gccgccggaa ttcgttcctc ctggctgcac ctgccgcagt gcacagtccg gctgaggtgc 5760 acgggagccc gccggcctct ctctgcccgc gtccgtccgt gaaattccgg ccggggctca 5820 ccgcgatggc cctcccgaca ccctcggaca gcaccctccc cgcggaagcc cggggacgag 5880 gacggccacg gagactcgtt tggaccccga gccaaagcga ggccctgcga gcctgctttg 5940 agcggaaccc gtacccgggc atcgccacca gagaacggct ggcccaggcc atcggcattc 6000 cggagcccag ggtccagatt tggtttcaga atgagaggtc acgccagctg aggcagcacc 6060 ggcgggaatc tcggccctgg cccgggagac gcggcccgcc agaaggccgg cgaaagcgga 6120 ccgccgtcac cggatcccag accgccctgc tcctccgagc ctttgagaag gatcgctttc 6180 caggcatcgc cgcccgggag gagctggcca gagagacggg cctcccggag tccaggattc 6240 agatctggtt tcagaatcga agggccaggc acccgggaca gggtggcagg gcgcccgcgc 6300 aggcaggcgg cctgtgcagc gcggcccccg gcgggggtca ccctgctccc tcgtgggtcg 6360 ccttcgccca caccggcgcg tggggaacgg ggcttcccgc accccacgtg ccctgcgcgc 6420 ctggggctct cccacagggg gctttcgtga gccaggcagc gagggccgcc cccgcgctgc 6480 agcccagcca ggccgcgccg gcagaggggg tctcccaacc tgccccggcg cgcggggatt 6540 tcgcctacgc cgccccggct cctccggagc cggggcgctc tcccaccctc aggctcctcg 6600 gtggcctccg cacccgggca aaagccggga ggaccgggac ccgcagcgcg acggcctgcc 6660 gggcccctgc gcggtggcac agcctgggcc cgctcaagcg gggccgcagg ccaaggggtg 6720 cttgcgccac ccacgtccca ggggagtccg tggtggggct ggggccgggg tccccaggtc 6780 gccggggcgg cgtgggaacc ccaagccggg gcagctccac ctccccagcc cgcgcccccg 6840 ggacgcctcc gcctccgcgc ggcaggggca gatgcaaggc atcccggcgc cctcccaggc 6900 gctccaggag ccggcgccct ggtctgcact cccctgcggc ctgctgctgg atgagctcct 6960 ggcgagcccg gagtttctgc agcaggcgca acctctccta gaaacggagg ccccggggga 7020 gctggaggaa caagctttgc aaagatggat aaagttttaa acagagagga atctttgcag 7080 ccttaagttt actccctatc agtgatagag aacgtatgaa gagtttactc cctatcagtg 7140 atagagaacg tatgcagact ttactcccta tcagtgatag agaacgtata aggagtttac 7200 tcctatcag tgatagagaa cgtatgacca gtttactccc tatcagtgat agagaacgta 7260 tctacagttt actccctatc agtgatagag aacgtatatc cagtttactc cctatcagtg 7320 atagagaacg tataagcttt aggcgtgtac ggtgggcgcc tataaaagca gagctcgttt 7380 agtgaaccgt cagatcgcct ggagcaattc cacaacactt ttgtcttata ccaactttcc 7440 gtaccacttc ctaccctcgt aaacagacgc gtggtgaccg ccgccaccat gggctgcatc 7500 aagagcaagc gcaaggacaa cctgaacgac gacggcgtgg acatggtgag caagggcgag 7560 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 7620 aagttcagcg tccgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag 7680 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc 7740 tacggcgtgg cctgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag 7800 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct ctttcaagga cgacggtacc 7860 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 7920 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc 7980 aacagccaca acgtctatat cacggccgac aagcagaaga acggcatcaa ggctaacttc 8040 aagatccgcc acaacgttga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 8100 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ccatcagtcc 8160 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtaacc 8220 gccgccggat ccggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa 8280 aaccccgggc ctatggaaga tgccaaaaac attaagaagg gcccagcgcc attctaccca 8340 ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca tgaagcgcta cgccctggtg 8400 cccggcacca tcgcctttac cgacgcacat atcgaggtgg acattaccta cgccgagtac 8460 ttcgagatga gcgttcggct ggcagaagct atgaagcgct atgggctgaa tacaaaccat 8520 cggatcgtgg tgtgcagcga gaatagcttg cagttcttca tgcccgtgtt gggtgccctg 8580 ttcatcggtg tggctgtggc cccagctaac gacatctaca acgagcgcga gctgctgaac 8640 agcatgggca tcagccagcc caccgtcgta ttcgtgagca agaaagggct gcaaaagatc 8700 ctcaacgtgc aaaagaagct accgatcata caaaagatca tcatcatgga tagcaagacc 8760 gactaccagg gcttccaaag catgtacacc ttcgtgactt cccatttgcc acccggcttc 8820 aacgagtacg acttcgtgcc cgagagcttc gaccgggaca aaaccatcgc cctgatcatg 8880 aacagtagtg gcagtaccgg attgcccaag ggcgtagccc taccgcaccg caccgcttgt 8940 gtccgattca gtcatgcccg cgaccccatc ttcggcaacc agatcatccc cgacaccgct 9000 atcctcagcg tggtgccatt tcaccacggc ttcggcatgt tcaccacgct gggctacttg 9060 atctgcggct ttcgggtcgt gctcatgtac cgcttcgagg aggagctatt cttgcgcagc 9120 ttgcaagact ataagattca atctgccctg ctggtgccca cactatttag cttcttcgct 9180 aagagcactc tcatcgacaa gtacgaccta agcaacttgc acgagatcgc cagcggcggg 9240 gcgccgctca gcaaggaggt aggtgaggcc gtggccaaac gcttccacct accaggcatc 9300 cgccagggct acggcctgac agaaaacc agcgccattc tgatcacccc cgaaggggac 9360 gacaagcctg gcgcagtagg caaggtggtg cccttcttcg aggctaaggt ggtggacttg 9420 gacaccggta agacactggg tgtgaaccag cgcggcgagc tgtgcgtccg tggccccatg 9480 atcatgagcg gctacgttaa caaccccgag gctacaaacg ctctcatcga caaggacgg 9540 tggctgcaca gcggcgacat cgcctactgg gacgaggacg agcacttctt catcgtggac 9600 cggctgaaga gcctgatcaa atacaagggc taccaggtag ccccagccga actggagagc 9660 atcctgctgc aacaccccaa catcttcgac gccggggtcg ccggcctgcc cgacgacgat 9720 gccggcgagc tgcccgccgc agtcgtcgtg ctggaacacg gtaaaaccat gaccgagaag 9780 gagatcgtgg actatgtggc cagccaggtt aaaccgcca agaagctgcg cggtggtgtt 9840 gtgttcgtgg acgaggtgcc taaggactg accggcaagt tggacgccccg caagatccgc 9900 gagattctca ttaaggccaa gaagggcggc aagatcgccg tgggatccgg aggatccgga 9960 gctacgaatt tcagtttgtt gaaacaagcg ggcgatgtcg aagaaaaccc cggccccatg aagttatggg atgtcgtggc tgtctgcctg gtgctgctcc acaccgcgtc cgccttcccg 10080 ctgcccgccg gtaagaggcc tcccgaggcg ccccccgag accgctccct cggccgccgc 10140 cgcgcgccct tcgcgctgag cagtgactca aatatgccag aggattatcc tgatcagttc gatgatgtca tggattttat tcaagccacc attaaagac tgaaaaggtc accagataaa caaatggcag tgcttcctag aagagagcgg aatcggcagg ctgcagctgc caacccagag 10380. aattccagag gaaaaggtcg gagaggccag aggggcaaaa accggggttg tgtcttaact gcaatacatt taaatgtcac tgacttgggt ctgggctatg aaaccaagga ggaactgatt tttaggtact gcagcggctc ttgcgatgca gctgagacaa cgtacgacaa aatattgaaa aacttatcca gaatagaag gctggtgagt gacaaagtag ggcaggcatg ttgcagaccc atcgcctttg atgatgacct gtcgttttta gatgataacc tggtttacca tattctaaga aagcattccg ctaaaaggtg tggatgtatc tgattcgaaa gccgcactcc tcataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt 10740 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct 10800 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 10860 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg 10920 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc 10980 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 11040 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt 11100 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca 11160 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt 11220 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcttaatta acttcgaact 11280 agggataaca gggtaatctc gactgtgcct tctagaaatt taaatcctcg agggccgcac 11340 tcctcagccg ctgtaagtct gcagaaattg atgatctatt aaaacataaa gatgtccact 11400 aaaatggaag ttttcctgt catactttgt taagaagggt gagacagag tacctacatt 11460 ttgaatggaa ggattggagc tacggggtg ggggtggggt gggattagat aaatgcctgc 11520 tctttactga aggctcttta ctattgcttt atgataatgt ttcatagttg gatatcataa 11580 tttaaaacaag caaaaccaaa ttaagggcca gctcattcct cccactcatg atctatagat 11640 ctatagatct ctcgtgggat cattgttttt ctcttgattc ccactttgtg gttctaagta 11700 ctgtggtttc caatgtgtc agttcatag cctgagaac gagatcagca gccctctgttc 11760 cacatacact tcattctcag tattgttttg ccaagttcta attchcatcag aagcttggat 11820 ccttagcga taagcttgat atcgaattcg agttggcgcg cctgtcattc taaatctctc 11880 ttcagccta aagctttc cccgtatccc cccaggtgtc tgcaggctca aagagcagcg 11940 agaagcgttc agaggaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca 12000 cgctgccggc tcgggatgc gggggaggcg ccggaccgga gcggagcccc gggcggctcg 12060 ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggagggggct 12120 gtccccgtga gctcccaggc gcgcctgtca ttctaaatct ctctttcagc ctaaagcttt 12180 ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa 12240 agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga 12300 tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc ccctagcgg 12360 gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagctcaat 12420 aaaagagccc acaacccctc actcggcgg ccagtcctc gatagactgc gtcgcccggg 12480 taccgtgta tccaataaac cctcttgcag cctgcaggaa gttcctattc tctagaaagt 12540 ataggaactt caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 12600 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 12660 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 12720 aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa 12780 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac 12840 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt 12900 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc 12960 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 13020 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg 13080 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 13140 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 13200 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 13260 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 13320 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 13380 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 13440 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 13500 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 13560 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 13620 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 13680 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 13740 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 13800 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 13860 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 13920 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 13980 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 14040 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 14100 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 14160 atatacga tacgggagg cttaccatct ggccccagtg ctgcaatgat accgcgagac 14220 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 14280 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 14340 Information gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 14400 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 14460 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttgg tcctccgatc 14520 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 14580 tctcttactg tcatgccatc cgtaagatgc tttctgtga ctggtgagta ctcaaccaag 14640 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 14700 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 14760 cgaaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 14820 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 14880 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 14940 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 15000 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 15060 ccacctg 15067

Claims

1. 1. A pharmaceutical for use in a method of increasing glial-derived neurotrophic factor (GDNF) levels in the central nervous system of an individual suffering from a neurodegenerative disorder, comprising a plurality of induced pluripotent stem cell-derived neural progenitor cells (iNPCs), the pharmaceutical comprising: The iNPC expresses an expression cassette integrated into its genome from a vector; The expression cassette is flanked by two transposon-containing elements contained in the vector, and A constitutive promoter operably linked to a tet responsive element; an inducible bidirectional polycistronic promoter comprising the tet response element; and GDNF coding sequence Including, The said medicine.

2. The pharmaceutical composition of claim 1, wherein the neurodegenerative disorder is selected from amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, and Alzheimer's disease.

3. the central nervous system includes regions of the brain; Optionally, said region of the brain comprises the substantia nigra, and / or the motor cortex, and / or the entorhinal cortex, and / or the hippocampus; Optionally, the individual is a human. The pharmaceutical composition according to claim 1 or 2.

4. the genomically integrated expression cassette is integrated into a region of the genome in which the probability of oncogenic transformation of a cell containing the genomically integrated expression cassette is reduced; and / or the genomically integrated expression cassette is integrated into a region of the genome selected from the adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, and the human orthologue of the mouse Rosa26 locus; and / or the expression cassette integrated into the genome is integrated by homologous recombination, and / or the expression cassette integrated into the genome is integrated as a single copy; and Optionally, a tetracycline class antibiotic is further administered, wherein said tetracycline class antibiotic induces transcription of GDNF mRNA in said plurality of induced pluripotent stem cell-derived neural progenitor cells. The pharmaceutical composition according to any one of claims 1 to 3.

5. 1. A medicament for use in a method of treating a neurodegenerative disease, comprising a quantity of induced pluripotent stem cell-derived neural progenitor cells (iNPCs), the cells inducibly expressing a neurotrophic factor capable of treating the disease, and The iNPC expresses an expression cassette integrated into the genome by nucleofection, the expression cassette being expressed from a vector and flanked by two transposon-containing elements contained in the vector; The expression cassette comprises: A constitutive promoter operably linked to a tet responsive element; an inducible bidirectional polycistronic promoter comprising the tet response element; and Glial-derived neurotrophic factor (GDNF) coding sequence Including, Optionally, a tetracycline, analog or derivative thereof is further administered. Medicine.

6. A quantity of neural progenitor cells capable of inducible expression of glial derived neurotrophic factor (GDNF), comprising a quantity of induced pluripotent stem cell derived neural progenitor cells (iNPCs), The iNPC expresses an expression cassette integrated into its genome from a vector; The expression cassette is flanked by two transposon-containing elements contained in the vector, and A constitutive promoter operably linked to a tet responsive element; an inducible bidirectional polycistronic promoter comprising the tet response element; and GDNF coding sequence Including, The neural progenitor cell.

7. A pharmaceutical for use in therapy comprising the cells described in claim 6.

8. A pharmaceutical for use in a method for treating amyotrophic lateral sclerosis (ALS), comprising the cells described in claim 6.

9. The pharmaceutical described in claim 1, wherein an iNPC expressing an expression cassette incorporated into the genome is generated by introducing the vector into an iNPC by one or more of nucleofection, transfection, and electroporation.

10. The pharmaceutical agent of claim 9, wherein the vector comprises the expression cassette and at least one homologous recombination sequence comprising a sequence capable of targeting one of the adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, and the human ortholog of the mouse Rosa26 locus, so that the expression cassette is integrated into a region of the genome selected from the group consisting of the adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, and the human ortholog of the mouse Rosa26 locus.

11. The pharmaceutical of claim 9, wherein GDNF secretion can be induced and regressed multiple times in iNPCs, and wherein transcription of GDNF mRNA is induced in multiple iNPCs in response to administration of tetracycline, an analog or derivative thereof, or doxycycline to an individual.

12. The pharmaceutical composition of claim 1, wherein addition of tetracycline, a tetracycline analog or derivative, or doxycycline to an individual causes the tet-On transactivator to bind to the inducible bidirectional polycistronic promoter and catalyzes transcription of an introduced gene downstream of the inducible bidirectional polycistronic promoter.

13. 1. A medicament for use in a method of treatment of a disease or condition comprising a quantity of cells, the cells expressing a therapeutic protein or peptide, the cells, the therapeutic protein or peptide, or both, capable of treating said disease or condition, and optionally the cell is a neural cell, optionally the neural cell is a neural progenitor cell, optionally the neural progenitor cell is derived from an induced pluripotent stem cell (iPSC); the cells express an expression cassette from one or more vectors; the expression cassette is flanked by two transposon-based elements contained in one of the one or more vectors; and A constitutive promoter operably linked to a tet responsive element; an inducible bidirectional polycistronic promoter comprising the tet response element; and A sequence encoding said therapeutic protein or peptide and The cells expressing the expression cassette are nucleofected, transfected, or electroporated. Medicine.

14. the one or more vectors comprise a piggyBac vector, a pBase vector, or both; Optionally, the piggyBac vector comprises an expression cassette. The pharmaceutical composition according to claim 13.

15. The expression cassette is integrated into a genome; Optionally, the expression cassette encodes the therapeutic protein or peptide, and / or the therapeutic protein or peptide comprises a neurotrophic factor, and / or the neurotrophic factor comprises glial derived neurotrophic factor (GDNF). The pharmaceutical composition according to claim 13.

16. the disease or condition is a neurodegenerative disease, Optionally, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), and / or the amount of cells is administered by injection, and / or tetracycline, an analog or derivative thereof is further administered. The pharmaceutical composition according to claim 13.

17. Providing a quantity of induced pluripotent stem cell (iPSC)-derived cells; and introducing at least two vectors into said iPSC-derived cells; A method comprising: the iPSC-derived cells are neural progenitor cells; and / or and / or wherein the introduction of the at least two vectors comprises one or more of nucleofection, transfection and electroporation; and / or the at least two vectors include a piggyBac vector and a pBase vector; Optionally, the piggyBac vector comprises: A constitutive promoter operably linked to a tet responsive element; an inducible bidirectional polycistronic promoter comprising the tet response element; and Protein or peptide coding sequences an expression cassette comprising Two transposon-containing elements contained in the piggyBac vector flank the expression cassette; and / or the protein or peptide comprises a neurotrophic factor, optionally the neurotrophic factor comprises glial derived neurotrophic factor (GDNF); and / or the piggyBac vector comprises a homologous recombination sequence; and / or the homologous recombination sequence comprises a sequence capable of targeting a genomic safe harbor; and / or The genomic safe harbor is one of the following: adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, human orthologue of mouse Rosa26 locus; method.

18. 20. A quantity of cells produced by the method of claim 17, wherein the iPSC-derived cells express an expression cassette integrated into the genome.

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