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  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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Definitions

• Described herein are cells engineered to express ectopic proteins in an inducible manner.
• the claimed invention relates to the technical field of regenerative medicine and degenerative diseases, including neurodegeneration.
• ALS Amyotrophic lateral sclerosis
• constitutive trophic factor release may be sufficient without considering any long term effects of continual administration of the drug.
• many other neurological diseases with a longer life span may not require sustained growth factor secretion, or it may be detrimental with chronic delivery.
• postmortem analysis of a Parkinsonian patient only temporarily receiving GDNF in the caudate putamen showed persistent neuronal sprouting that may explain maintained effects after GDNF delivery cessation.
• constitutive growth factor release does not allow for cessation in the event of a contraindication. For instance, trials have reported that some patients receiving GDNF developed Lhermitte’s symptoms.
• sustained GDNF expression can cause aberrant sprouting and desensitization of the targeted neurons. Importantly, these effects were reversed upon GDNF cessation. Overall, there is a clear rationale for growth factor regulation in these types of clinical applications and a great need in the art for compositions and techniques achieving these aims.
• Combined cell and gene therapy approaches to both rejuvenate cellular niches and provide therapeutic molecules to diseased host cells is a promising new treatment approach for neurogical disorders.
• the Inventors’ 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. Critically, these cells have been confirmed to be safe and non-tumorigenic and as such are now being used in the first-ever cell and gene therapy FDA-approved Phase l/2a clinical trial for the protection of motor neurons in ALS patients.
• GDNF tetracycline
• iPSC human induced pluripotent stem cell
• iNPCs human induced pluripotent stem cell
• dox administration can inducibly and reversibly modulate GDNF secretion in vivo.
• the Inventors demonstrate that the powerful technologies of iPSCs, ex vivo cell engineering, and gene regulation can be combined as a unique approach to treat disorders where regulated protein delivery may be desired.
• Described herein is a method of treatment, including administering a quantity of cells to a subject afflicted with a disease or condition, wherein the cells express a therapeutic protein or peptide, and further wherein the cells, therapeutic protein or peptide, or both, are capable of treating the disease or condition.
• the cells are neural lineage cells.
• the neural lineage cells are neural progenitor cells.
• the neural progenitor cells are derived from induced pluripotent stem cells (iPSCs).
• the cells express an expression cassette from one or more vectors.
• the cells expressing the expression cassette from the one or more vectors have been nucleofected, transfected, or electroporated.
• the one or more vectors comprise a piggyBac vector, a pBase vector, or both.
• the piggyBac vector includes at least two promoters, wherein at least one promoter is inducible.
• the at least one inducible promoter is polycistronic.
• the at least one inducible, polycistronic promoter is bi- directional.
• the expression cassette is genomically integrated.
• the expression cassette encodes the therapeutic protein or peptide.
• the therapeutic protein or peptide includes a neurotrophic factor.
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF).
• the disease or condition is a neurodegenerative disease.
• the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
• administering a quantity of cells includes injection.
• the method includes administration of tetracycline, an analog or derivative thereof, including for example, doxycycline.
• Also described herein is a method, including providing a quantity of induced pluripotent stem cell (iPSC) derived cells and introducing at least two vectors into the iPSC derived cells.
• the iPSC derived cells are neural progenitor cells.
• introducing at least two vector includes one or more of: nucleofection, transfection and electroporation.
• the at least two vectors comprise a piggyBac vector and a pBase vector.
• the piggyBac vector includes an expression cassette, including a constitutive promoter, an inducible promoter including a tet responsive element, and a sequence encoding a protein or peptide, two transposon elements, wherein the two transposon elements flank the expression cassette.
• the inducible promoter is a bi-directional polycistronic promoter.
• the protein or peptide includes a neurotrophic factor.
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF). Further described herein is a a quantity of cells made by the aforementioned method, wherein the iPSC derived cells express a genomically integrated expression cassette.
• GDNF glial derived neurotrophic factor
• iNPCs induced pluripotent stem cell derived neural progenitor cells
• the iNPCs express a genomically integrated expression cassette introduced by nucleofection, the expression cassette including a constitutive promoter, an inducible promoter including a tet response element, and a sequence encoding glial derived neurotrophic factor (GDNF).
• the inducible promoter is a bi-directional polycistronic promoter.
• the method includes administration of tetracycline, an analog or derivative thereof, including for example, doxycycline.
• a quantity of neural progenitor cells capable of inducible expression of glial derived neurotrophic factor (GDNF) including a quantity of induced pluripotent stem cell derived neural progenitor cells (iNPCs), wherein the iNPCs express a genomically integrated expression cassette including a constitutive promoter, an inducible promoter including a tet response element, and a sequence encoding GDNF.
• the inducible promoter is a bi-directional polycistronic promoter.
• ALS amyotrophic lateral sclerosis
• the genomically integrated expression cassette lacks a transposon derived sequence within lOkb of the genomically integrated expression cassette. In various embodiments, the genomically integrated expression cassette lacks a transposon derived sequence within lkb of the genomically integrated expression cassette. In various embodiments, the genomically integrated expression cassette lacks a viral derived sequence within lOkb of the genomically integrated expression cassette. In various embodiments, the genomically integrated expression cassette lacks a viral derived sequence within lkb of the genomically integrated expression cassette. Also described herein is a method of increasing glial derived neurotrophic factor
• GDNF induced pluripotent stem cell derived neural progenitor cells
• iNPCs induced pluripotent stem cell derived neural progenitor cells
• the neurodegenerative disorder is selected from amyotrophic lateral sclerosis (ALS), Parkinson’s, Huntington’s, and Alzheimer’s Diseases.
• the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS).
• the neurodegenerative disorder is Parkinson’s disease. In various embodiments, the neurodegenerative disorder is Alzheimer’s disease.
• the central nervous system includes a region of the brain. In various embodiments, the region of the brain includes the substantia nigra. In various embodiments, the region of the brain includes the motor cortex. In various embodiments, the region of the brain includes the entorhinal cortex and/or the hippocampus. In various embodiments, the individual is a human.
• the genomically integrated expression cassette is integrated at a region of the genome with a reduced probability of oncogenic transformation for a cell comprising the genomically integrated expression cassette.
• the genomically integrated expression cassette is integrated at a region of the genome selected from the adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, and a human ortholog of the mouse Rosa26 locus.
• the genomically integrated expression cassette is integrated at the adeno- associated virus site 1 (AAVS1).
• the genomically integrated expression cassette is integrated at the chemokine (C-C motif) receptor 5 (CCR5) gene.
• the genomically integrated expression cassette is integrated by homologous recombination.
• the genomically integrated expression cassette lacks sequences derived from a transposable element.
• the genomically integrated expression cassette is integrated by a transposon.
• the method further includes administering to the individual a tetracycline-class antibiotic, wherein the tetracycline-class antibiotic induces transcription of a GDNF mRNA in the plurality of induced pluripotent stem cell derived neural progenitor cells.
• the genomically integrated expression cassette is integrated as a single-copy.
• iNPCs induced pluripotent stem cell derived neural progenitor cells
• GDNF glial derived neurotrophic factor
• 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.
• the neurodegenerative disorder is selected from amyotrophic lateral sclerosis (ALS), Parkinson’s, Huntington’s, and Alzheimer’s Diseases.
• 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.
• the central nervous system includes a region of the brain. In various embodiments, the region of the brain includes the substantia nigra. In various embodiments, the region of the brain includes the motor cortex. In various embodiments, the region of the brain includes the entorhinal cortex and/or the hippocampus. In various embodiments, the individual is a human. In various embodiments, the genomically integrated expression cassette is integrated at a region of the genome with a reduced probability of oncogenic transformation for a cell comprising the genomically integrated expression cassette.
• the genomically integrated expression cassette is integrated at a region of the genome selected from the adeno- associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, and a human ortholog of the mouse Rosa26 locus.
• the genomically integrated expression cassette is integrated at the adeno-associated virus site 1 (AAVS1).
• the genomically integrated expression cassette is integrated at the chemokine (C-C motif) receptor 5 (CCR5) gene.
• the genomically integrated expression cassette is integrated by homologous recombination.
• the genomically integrated expression cassette lacks sequences derived from a transposable element.
• the genomically integrated expression cassette is integrated by a transposon.
• the genomically integrated expression cassette is integrated as a single-copy.
• Reporter transgene expression is inducible and reversible in iPSC- derived NPC transplants.
• IPSC-derived NPCs nucleofected with pB-RTP-Tet-GDNF/memClover- FLuc retain their ability to be induced upon the addition of dox. 83% of BFP + cells were also GFP + (P5 Gate) 24 hours after adding dox to cells that were grown in culture for 6 months after nucleofection. Note: Cells were also exposed to two freeze/thaw cycles and were frozen for a total of 10 months during this period.
• Figure 8 Vector map of AAVSl-teton-hGDNF. Shown are HA-L and HA-R arms, which are homologous recombination sequences that can be used to target genomic safe harbors, such as AAVS.
• Figure 10 Vector map of pDonor-Teton3g-2a-TagBFP-V5-nls-p2a-puroR WPRE Insulated mpclover-2a-luc2pest-2a-gdnf wpre.
• Figure 11 (A) Schematic of AAVS1 targeting of the endogenous locus between exon 1 and 2 of the human PPP1R12C gene.
• a recipient“landing site” consisting of a reporter/selection cassette (TagBFP2 and PuroR for fluorescent 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 a LoxP and an FRT site were stable integrated.
• these cells were lipofected with a plasmid expressing FlpO and Cre and the donor plasmid containing a LoxP and FRT flanked selection/reporter cassette and a dox-inducible mpClover/Luc2p/GDNF cistron-containing plasmid.
• control elements refers collectively 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 a coding sequence in a 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.
• promoter region is used herein in its ordinary sense to refer to a nucleotide region including a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3’ -directi on) coding sequence.
• operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
• control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
• the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
• intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered“operably linked” to the coding sequence.
• GDNF glial cell line-derived neurotrophic factor
• Tet tetracycline-regulated systems
• dox doxycycline
• Tet-Off a“Tet- On” system uses a reverse tTA (rtTA) in order to activate transgene expression in the presence of dox
• rtTA reverse tTA
• Described herein is a stably integrating, third-generation doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule.
• Human iPSC- derived neural progenitors were stably transfected with the vector, expanded and transplanted into the adult mouse brain.
• Our findings provide a proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.
• Described herein is a method of treatment, including administering a quantity of cells to a subject afflicted with a disease or condition, wherein the cells express a therapeutic protein or peptide, and further wherein the cells, therapeutic protein or peptide, or both, are capable of treating the disease or condition.
• the cells are neural lineage cells.
• the neural lineage cells are neural progenitor cells.
• the neural progenitor cells are derived from induced pluripotent stem cells (iPSCs).
• iPSCs induced pluripotent stem cells
• neural progenitor cells are generated from iPSC colonies by culturing in a neural stem cell medium containing high concentrations of EGF and FGF-2.
• EZ spheres Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact.
• EZ spheres are withdrawn from EGF/FGF, cultured with retinoic acid RA in a neural induction.
• Techniques related 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.
• the iPSCs include somatic cells such as fibroblasts and blood reprogrammed according to methods described in Ei.S. Pub. No. 2017/0362574, which is fully incorporated by reference herein.
• the cells express an expression cassette from one or more vectors.
• the cells expressing the expression cassette from the one or more vectors have been nucleofected, transfected, or electroporated or other gene delivery techniques known in the art.
• the one or more vectors includes a piggyBac vector, a pBase vector, or both.
• the piggyBac vector includes at least two promoters, wherein at least one promoter is inducible.
• the least one inducible promoter is polycistronic.
• the at least one inducible, polycistronic promoter is bi-directional.
• the expression cassette is genomically integrated.
• the expression cassette encodes the therapeutic protein or peptide.
• the therapeutic protein or peptide includes a neurotrophic factor.
• the one or more vectors include a vector with a gene expression cassette flanked by two transposon elements.
• the two transposon elements include piggyBac terminal repeats (PB TR).
• the vector includes the constitutive promoter includes CMV/Chick b-Actin (aka CAG) promoter.
• the vector includes an includible, bi-directional promoter includes TRE-Bi promoter.
• the constitutive promoter is operatively linked to a tet response elements.
• the“tet-on” element including for example, rTA.
• rTA includes rtTA-VlO.
• the constitutive promoter is operatively linked to a selection factor, including for example neomycin or puromycin.
• the inducible, bi-directional promoter is polycistronic.
• the inducible bi-directional promoter is operatively linked to elements in a first, second or third or more cistrons.
• a first, second, or third, or more cistrons includes a transgene.
• the transgene is followed by one or more post-transcriptional elements.
• the one or more post-transcriptional element includes woodchuck hepatitis virus post- transcriptional element (WPRE).
• the transgene is followed by one or more poly-A tails. In this includes, for example, rabbit beta-globin poly As.
• the transgene is a neurotrophic factor.
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF). Additional information is found in PCT Pub. No. WO 2017/131926 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 is fully incorporated by reference herein.
• the one or more vectors include a vector encoding a recombinase including VCre (Vlox and derivatives), SCre (Slox and derivatives), Dre (Rox and derivatives), and phiC31 (attb) or other recombinases known in the art.
• VCre Vlox and derivatives
• SCre Slox and derivatives
• Dre Rox and derivatives
• phiC31 attb
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF).
• the disease or condition is a neurodegenerative disease.
• the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
• administering a quantity of cells includes injection.
• the method includes administration of tetracycline, an analog or derivative thereof, including for example, doxycycline.
• the vector includes at least one homologous recombination sequence.
• the homologous recombination sequence includes a sequence capable of targeting a genomic safe harbor.
• the genomic safe harbor is one of: the adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, human ortholog of the mouse Rosa26 locus.
• An exemplary sequence of one of the aforementioned vectors is SEQ ID NO: 2 and SEQ ID NO: 3.
• iPSC induced pluripotent stem cell
• introducing at least two vectors includes one or more of: nucleofection, transfection and electroporation.
• the at least two vectors includes a piggyBac vector and a pBase vector.
• the piggyBac vector includes an expression cassette, including a constitutive promoter, an inducible, bi-directional polycistronic promoter including a tet responsive element, and a sequence encoding a protein or peptide, two transposon elements, wherein the two transposon elements flank the expression cassette.
• the protein or peptide includes a neurotrophic factor.
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF).
• the vector includes at least one homologous recombination sequence.
• the vector and/or cells comprising the vector lack a transposable element and/or a transposon derived sequence within 500bp, 1 kb, 2kb, 5kb, or 10 kb of the expression cassette. In various embodiments, the vector and/or cells comprising the vector lack a virally derived sequnce within 500bp, 1 kb, 2kb, 5kb, or 10 kb of the expression cassette. In various embodiments, the cells includes 1 or 2 copies of the genomically integrated vector. In various embodiments, the cells includes the vector genomically integrated as a single copy.
• a quantity of cells made by the aforementioned method wherein the iPSC derived cells express a genomically integrated expression cassette.
• a quantity of neural progenitor cells capable of inducible expression of glial derived neurotrophic factor (GDNF) made by a method including providing a quantity of induced pluripotent stem cell derived neural progenitor cells (iNPCs), and introducing a piggyBac vector and a pBase vector into the iNPCs derived cells, wherein the piggyBac vector includes a constitutive promoter, an inducible, bi-directional polycistronic promoter including a tet response element, and a sequence encoding GDNF.
• the vector includes at least one homologous recombination sequence.
• a method of treating a degenerative disease and/or condition using the aforementioned cells including a neurodegenerative disease such as amyotrophic lateral sclerosis (ALS).
• a neurodegenerative disease such as amyotrophic lateral sclerosis (ALS).
• iNPCs induced pluripotent stem cell derived neural progenitor cells
• the iNPCs express a genomically integrated expression cassette introduced by nucleofection, the expression cassette including a constitutive promoter, an inducible, bi-directional polycistronic promoter including a tet response element, and a sequence encoding glial derived neurotrophic factor (GDNF).
• the vector includes at least one homologous recombination sequence.
• the method includes administration of tetracycline, an analog or derivative thereof.
• a quantity of neural progenitor cells capable of inducible expression of glial derived neurotrophic factor (GDNF), wherein the neural progenitor cells include a genomically integrated expression cassette, including a bi-directional polycistronic promoter including a tet response element, and a sequence encoding GDNF.
• a method of treating a degenerative disease and/or condition using the aforementioned cells including a neurodegenerative disease such as amyotrophic lateral sclerosis (ALS).
• ALS amyotrophic lateral sclerosis
• a vector including a gene expression cassette including a constitutive promoter, an inducible, bi-directional promoter including a tet response element, and a sequence encoding a protein or peptide.
• the gene expression cassette is flanked by two transposon elements.
• the two transposon elements include piggyBac terminal repeats (PB TR).
• the two transposon elements include loxP and flippase recognition target, or other transposon elements known in the art.
• the constitutive promoter includes CMV/Chick b-Actin (aka CAG) promoter.
• the includible, bi- directional promoter includes TRE-Bi promoter.
• the constitute promoter is operatively linked to a tet response elements.
• the tet response element is a“tet-off” element, including for example, tTA, or a“tet-on” element including for example, rTA.
• rTA includes rtTA-VlO.
• the constitutive promoter is operatively linked to a selection factor, including for example neomycin or puromycin.
• the inducible, bi-directional promoter is polycistronic.
• the inducible bi-directional promoter is operatively linked to elements in a first, second or third or more cistrons.
• a first, second, or third, or more cistrons includes a transgene.
• the transgene is followed by one or more post-transcriptional elements.
• the one or more post-transcriptional element includes woodchuck hepatitis virus post-transcriptional element (WPRE).
• WPRE woodchuck hepatitis virus post-transcriptional element
• the transgene is followed by one or more poly-A tails. In this includes, for example, rabbit beta-globin polyAs.
• the transgene is a neurotrophic factor.
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF).
• the vector includes one or more elements promoting target of safe landing sites, including AAVS1.
• the expression cassette includes one or more sub-cassettes, wherein each sub- cassette includes 1) a promoter 2) a transgene and 3) a polyA transcription stop element.
• the expression cassette including one or more sub-cassettes includes a constitutive sub-cassette and an inducible sub-cassette.
• the constitutive sub- cassette includes the constitutive promoter expressing rTA transactivator
• the inducible sub-cassette includes an inducible promoter expressing a neurotrophic factor such as GDNF and optionally one or more reporter proteins.
• an example of the aforementioned vector includes pB-RTP-Tet-GDNF/memClover- FLuc” [pi ggyBac- Reverse transactivator/TagBFP2nls/PacR-Tet inducible-GDNF/membrane Clover-Firefly Luciferase] [SEQ ID NO: 1], which is depicted in Figure 1A.
• the vector includes two promoters - a constitutively active CMV/Chick b-Actin (aka CAG) promoter and an inducible, bi-directional TRE-Bi promoter.
• the CAG promoter drives constitutive expression of the rtTA-VlO (aka tet-ON) transactivator, TagBFP2-V5nls (enhanced blue fluorescent protein with a V5 tag and nuclear localization sequence), and the puromycin resistance gene.
• Transgenes in tandem are separated by self-cleaving peptide linkers (P2A).
• P2A self-cleaving peptide linkers
• addition of a tetracycline analog or derivative, doxy cy cline causes the rtTA-VlO transactivator to bind to the TRE-Bi promoter and catalyze transcription of downstream transgenes.
• the first cistron of the TRE-Bi promoter harbors a myristoylated and palmitoylated (MyrPalm) clover reporter (mpClover) followed by destabilized firefly luciferase (Luc2P).
• MyrPalm myristoylated and palmitoylated clover reporter
• Luc2P destabilized firefly luciferase
• the second cistron downstream of the inducible TRE-Bi promoter can encode a neurotrophic factor such as GDNF followed by the woodchuck hepatitis virus post- transcriptional element (WPRE) for increased gene expression.
• WPRE woodchuck hepatitis virus post- transcriptional element
• the pB-RTP-Tet-GDNF/memClover-FLuc vector can be transfected alongside a pBase plasmid to promote stable genomic integration.
• Another example of the aforementioned vector includes pDonor-Teton3g-2a-TagBFP-V5-nls-p2a- puroR WPRE Insulated mpclover-2a-luc2pest-2a-gdnf wpre shown in Fig. 10.
• the vector includes at least one homologous recombination sequence.
• the homologous recombination sequence includes a sequence capable of targeting a genomic safe harbor.
• the genomic safe harbor is one of: the adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, human ortholog of the mouse Rosa26 locus.
• An exemplary sequence of one of the aforementioned vectors is SEQ ID NO: 2 and SEQ ID NO: 3.
• Described herein is a method of treatment, comprising: administering a quantity of cells to a subject afflicted with a disease or condition, wherein the cells express a therapeutic protein or peptide, and further wherein the cells, therapeutic protein or peptide, or both, are capable of treating the disease or condition.
• the cells are neural lineage cells.
• the neural lineage cells are neural progenitor cells.
• the neural progenitor cells are derived from induced pluripotent stem cells (iPSCs).
• the cells express an expression cassette from one or more vectors.
• the cells expressing the expression cassette from the one or more vectors have been nucleofected, transfected, or electroporated.
• the one or more vectors includes a piggyBac vector, a pBase vector, or both.
• the piggyBac vector includes at least two promoters, wherein at least one promoter is inducible.
• the at least one inducible promoter is polycistronic.
• the at least one inducible, polycistronic promoter is bi-directional.
• the expression cassette is genomically integrated.
• the expression cassette encodes the therapeutic protein or peptide.
• the therapeutic protein or peptide includes a neurotrophic factor.
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF).
• the disease or condition is a neurodegenerative disease.
• the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
• administering a quantity of cells includes injection.
• the method includes administration of tetracycline, an analog or derivative thereof.
• Described herein is a method comprising: providing a quantity of induced pluripotent stem cell (iPSC) derived cells; and introducing at least two vectors into the iPSC derived cells.
• the iPSC derived cells are neural progenitor cells.
• introducing at least two vector includes one or more of: nucleofection, transfection and electroporation.
• the at least two vectors includes a piggyBac vector and a pBase vector.
• the piggyBac vector includes: an expression cassette, comprising: a constitutive promoter, an inducible, bi-directional polycistronic promoter comprising a tet responsive element, and a sequence encoding a protein or peptide, two transposon elements, wherein the two transposon elements flank the expression cassette.
• protein or peptide includes a neurotrophic factor.
• the neurotrophic factor includes glial derived neurotrophic factor (GDNF).
• the piggyBac vector includes a homologous recombination sequence.
• the homologous recombination sequence includes a sequence capable of targeting a genomic safe harbor.
• the genomic safe harbor is one of: the adeno-associated virus site 1 (AAVS1), the chemokine (C-C motif) receptor 5 (CCR5) gene, human ortholog of the mouse Rosa26 locus.
• iNPCs induced pluripotent stem cell derived neural progenitor cells
• iNPCs express a genomically integrated expression cassette introduced by nucleofection, the expression cassette comprising: a constitutive promoter, an inducible, bi-directional polycistronic promoter comprising a tet response element, and a sequence encoding glial derived neurotrophic factor (GDNF).
• the method includes administration of tetracycline, an analog or derivative thereof.
• dox solution was maintained in light protection and 4°C.
• dox (Clontech 631311) was added to culture media at lOOng/ml.
• animals were administered dox (15 pg dox/g weight) every 3-4 days by oral gavage (e.g. 60m1 for a 20g animal), using a soft-tipped feeding needle (Instech FTP-20-30; Madison Meeting, PA) attached to a lml syringe (BD 309659; Franklin Lakes, NJ).
• cells such as fibroblasts are reprogrammed into virus-free iPSC lines using the Amaxa Human Dermal Fibroblast Nucleofector Kit to express episomal plasmids with 6 factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA (Addgene).
• Exogenously introduced genes do not integrate and are instead expressed episomally in a transient fashion.
• fibroblasts 0.8 c 106 cells per nucleofection
• the small molecules used werel) sodium butyrate (0.5 mM), 2) glycogen synthase kinase 3b inhibitor of the Wnt/p-catenin signaling pathway (CHIR99021, 3 mM), 3) MEK pathway inhibitor (PD 0325901, 0.5 mM), 4) Selective inhibitor of TGF-b type I receptor ALK5 kinase, type I activin/nodal receptor ALK4 and type I nodal receptor ALK7 (A 83-01, 0.5 pM). Colonies with ES/iPSC-like morphology appeared 25-31 days later. Subsequently, colonies with the best morphology were transferred onto a feeder-independent 6 BD MatrigelTM Matrix and maintained in mTeSR®l medium. The iPSC clones were further expanded and cryopreserved.
• EZ spheres generated from iPSCs can be differentiated to a culture of neural progenitor cells in suspension (iNPCsSU), with astroglial predisposition.
• iNPCsSU neural progenitor cells in suspension
• EZ spheres were caudalized using retinoic acid RA (0.5pM) in Neural Induction Media (NIM) (DMEM/F12, 1% NEAA, 1% N2, heparin 2pg/ml; Sigma). This media 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).
• NAM Neural Induction Media
• iNPCsSU maintain their proliferative potential and astroglial generation propensity for 26-30 passages and can be efficiently cryopreserved.
• iNPCsAD an adherent format of iNPCs grown as adherent cultures and 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).
• iNPCsSU were dissociated to 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.
• Nucleofected iNPCs which underwent two rounds of FACS for BFP, were transplanted into the striatum (2m1 of cells at 100,000 cells/m ⁇ into each hemisphere) of 6- month old NOD-SCID mice (Nod.cbl7-Prkdc scld /J, Jackson Lab #1303; Bar Harbor, ME). Three days before transplantation, a subset of cells was treated with dox. For transplantation, cells were dissociated and diluted to a final concentration of 100,000 cells/pl in conditioned media, and maintained on ice. Animals were anesthetized with isoflurane and placed in a stereotaxic frame.
• Cells were injected bilaterally relative to bregma at 0.7mm rostral and +/- 2.0mm lateral.
• a 5m1 Hamilton microsyringe backfilled with PBS was loaded with 2m1 of cell solution and inserted to a depth of -4.00mm.
• the microsyringe was then raised to -3.50mm and the cell solution was injected at lpl/min. After cell injection, the microsyringe was raised to -3.00mm, held for 2 minutes and slowly withdrawn. Animals were checked daily.
• Tet-ON systems rely on constitutive expression of the rtTA-VlO transactivator and a dox-responsive TRE-Bi (Tet-Response Element Bi-directional) promoter.
• the Inventors observed that if the transactivator and inducible promoter are expressed in different plasmids and co-transfected into cells, the cells drifted during expansion and selected against doubly- transgenic populations, resulting in cells that are non-inducible (data not shown).
• the vector has two promoters - a constitutively active CMV/Chick b-Actin (aka CAG) promoter and an inducible, bi-directional TRE-Bi promoter.
• the CAG promoter ( Figure 1A 2 ) drives constitutive expression of the rtTA-VlO (aka tet-ON) transactivator ( Figure 1A 3 ), TagBFP2-V5nls (enhanced blue fluorescent protein with a V5 tag and nuclear localization sequence) ( Figure 1A 4 ), and the puromycin resistance gene (Figure lAs).
• Transgenes in tandem are separated by self-cleaving peptide linkers (P2A).
• Clover is a green fluorescent protein, which the MyrPalm sequences localizes to the cell membrane.
• a membrane fluorescent reporter was chosen in order to avoid visual overlap with perinuclear GDNF localization.
• Destabilized firefly luciferase (Luc2P) which has a half-life several folds less than wild-type luciferase, was used for analysis of gene expression in live animals over time.
• the second cistron downstream of the inducible TRE-Bi promoter harbors GDNF ( Figure lAn) followed by the woodchuck hepatitis virus post-transcriptional element (WPRE) for increased gene expression ( Figure IA 12 ).
• Immunocytochemistry was performed as previously described. Briefly, cells were fixed in 4% paraformaldehyde (PFA) for 12 minutes. The PFA solution was then removed and washed three times with Phosphate Buffered Saline (PBS) with a 5-minute incubation each. Cells were then incubated in primary antibodies [concentrations listed in Table 1] diluted in PBS with 0.3% Triton (PBS-T) and 3% normal donkey serum (NDS) for at least 12 hours at 4°C on a gentle rocker, followed by three 5 min PBS washes at room temperature.
• PFA paraformaldehyde
• PBS Phosphate Buffered Saline
• Media used for ELISA was devoid of EGF and FGF and composed of DMEM:Fl2 (70:30) supplemented with 1% Antibiotic-Antimycotic and 2% B27 without Vitamin A. Before media was added for an incubation period of 24 hours or 7 days, old media was aspirated and wells were washed twice with sterile PBS. Collected media was stored at - 80°C. A GDNF ELISA was performed per manufacturer instructions (R&D Systems DY212;
• confocal images were collected on a Nikon A1R inverted laser scanning confocal microscope with appropriate settings to sequentially image colors and avoid signal crosstalk. The exposure and saturation measures were utilized to capture the maximum dynamic range. Typically, after the exposure was set, the identical setting was reused for the subsequent samples in the group.
• ND2 image files were initially imported into ImageJ for manipulation of confocal Z- stacks or for isolation of individual channels from single z-slices for subsequent editing in Adobe Photoshop CS6. Image curves were adjusted for consistency of dynamic range and exposure in Photoshop CS6, cropped, and then imported into Adobe Illustrator CS6 for the preparation of final images.
• Reporter protein production is reversibly regulated in vitro
• iNPCs were nucleofected as above, along with an Efl-renilla plasmid that constitutively expresses renilla luciferase for protein normalization.
• a dual-luciferase assay revealed that cells expressed a ⁇ l 300-fold increase in firefly luciferase activity (normalized to renilla) after a 24-hour dox treatment, and this returned to near basal levels upon dox withdrawal (Figure IE).
• GDNF production was next assessed in nucleofected iNPCs. Immunofluorescence revealed that GDNF was not visible in cells in the absence of dox, which confirms the Inventors’ previous report that wildtype 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 the constitutive promoter ( Figure 2A). In the presence of dox, GDNF as well as Clover and luciferase were detected, along with maintained BFP production ( Figure 2B). When dox was subsequently removed, GDNF and reporter gene production all ceased, while BFP was maintained ( Figure 2C).
• Reporter transgene expression is inducible and reversible in iNPCs transplants in the adult brain
• nucleofected iNPCs were expanded and transplanted to the adult NOD-SCID mouse brain ( Figure 3A).
• the parent cell population contained 15.6% BFP + , which were then expanded as neurospheres for 7-weeks and sorted a second time for BFP, which yielded 32.4% BFP + cells.
• GDNF expression is inducible and reversible in iNPC transplants
• Str Striatum.
• CC Corpus Callosum This analysis also revealed that the variations in bioluminescence intensity may be a result of varying graft location. Specifically, some animals that exhibited remarkably high bioluminescence signal had grafted cells in the meninges, which is likely due to cell reflux into the injection tract during needle retraction ( Figures 7). Overall, the extensive analysis of all study animals confirmed a strong correlation between bioluminescence signal and GDNF expression.
• GDNF produced by engineered human neural progenitor cells can be tightly regulated across multiple cycles in vivo. This technology can be applied to other growth factors, providing a valuable means to protect neurons damaged in different disease.
• dox penetrates the parenchymal transplant site to a level that allows for transgene activation from the inducible TRE-Bi promoter.
• transgene activity can be induced and reversed multiple cycles.
• GDNF administration can be attenuated or stopped if patients develop side-effects, desensitization, or other transgene-related phenomena during disease progression.
• the drug regimen could be varied to allow for re-sensitization of host receptors and signaling pathways. This is in stark contrast to ex vivo gene therapy with constitutive gene expression.
• the amount of dox concentration required for gene control (15 pg dox/g weight) is below antimicrobial doses and does not increase the presence of dox-resistant bacteria or negatively affect the gut flora.
• the novel dox-catalyzed system provides nearly two-fold higher GDNF secretion. This increase may be attributed to piggyBac transposition that can mediate more copies of plasmid genomic insertion than lentiviral transduction. Furthermore, GDNF transcription with previous lentiviral experiments is dependent on the PGK promoter that is activated by endogenous transcription factors. In the present study, GDNF transcription is initiated by binding of the tet-ON transactivator to the TRE-Bi promoter that is catalyzed by a conformational change initiated by dox administration.
• transcription is not limited by a naturally occurring transcription factor, but rather the amount of transactivator and dox present.
• transplanting ex vivo genetically engineered neural progenitors as done in this study adds a level of safety compared to direct genetic manipulation of host cells by viral transduction of pB-RTP-Tet-GDNF/memClover-Flu.
• direct viral transduction to the brain may not only target healthy cells neighboring diseased cells but also may further compromise diseased host cells.
• simply introducing healthy neural progenitor/stem cells, in it of itself may have beneficial effects on the diseased host mileu in various diseases. Therefore, the synergistic effect of both cell and inducible gene therapy may surpass gene therapy alone.
• the neural cells are derived from a human iPSC source, critically providing the promise of autologous transplantation.
• pBase mediates random integration
• the Inventors have not seen tumors in over 500 mice where pBase was used.
• a clinical grade product may necessitate single-copy site-specific integration.
• targeting the AAVS1 safe landing may allow for long-term stable integration and methylation-resistant expression of transgenes.
• the Inventors have observed marked dox-mediated transgene expression at the single-copy level using the same TRE-Bi promoter used in this study.
• AAVS1-AVPT This new intermediate vector is named AAVS1-AVPT.
• Intermediate vector AAVS1-AVPT and pB-RTP-Tet-GDNF/memClover-FLuc are digested with the restriction enzymes Sall and Sfil. The appropriate fragments were isolated and joined by DNA ligation. The resulting construct was confirmed by DNA sequencing and provisionally named AAVSl-teton-hGDNF. [SEQ ID NO: 2] as shown in Figure 8.
• the AAVSl-teton-hGDNF construct was introduced into the human iPSC line CS0002iCTR-nl 1 by electroporation.
• hAAVSl 1R TALEN was a gift from Feng Zhang (Addgene plasmid # 35432; http://n2t.net/addgene:35432; RRID: Addgene_35432)
• hAAVSl 1L TALEN was a gift from Feng Zhang (Addgene plasmid # 35431; http://n2t.net/addgene:3543 l; RRID :Addgene_35431)).
• Cultures are maintained in the presence of the tetracycline analog doxycycline to facilitate identification of AAVSl-teton-hGDNF containing cells based on the expression of the fluorescent protein Clover. “Green” colonies were handpicked under fluorescent microscopy for expansion. Once sufficient numbers of cells were produced the clones were further purified by FACS sorting. Simultaneously, culture media was collected from these clones to confirm GDNF production by ELISA assay.
• Such vectors can include reports such as eGFP and Luc2.
• An example includes pDonor-Teton3g-2a-TagBFP-V5-nls- p2a-puroR WPRE Insulated mpclover-2a-luc2pest-2a-gdnf wpre [SEQ ID NO: 3] as shown in Figure 10.
• Such vector includes homologous recombination sequences.
• AAVS1 targeting of the endogenous locus is between exon 1 and 2 of the human PPP1R12C gene.
• a recipient“landing site” includes a reporter/selection cassette (TagBFP2 and PuroR for fluorescent 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 a LoxP and an FRT site were stable integrated. Subsequently, upon stable selection for these reporters, these cells were lipofected with a plasmid expressing FlpO and Cre and the donor plasmid containing a LoxP and FRT flanked selection/reporter cassette and a dox-inducible mpClover/Luc2p/GDNF cistron-containing plasmid.
• a reporter/selection cassette (TagBFP2 and PuroR for fluorescent and antibiotic selection) driven by a splice acceptor linked to the upstream PPP1R12C and a constitutive CAG promoter driven td-Tomato red fluorescent cistron flanke
• Transmitted light and green fluorescence imaging of cells in the absence and presence of doxycycline demonstrates the inducibility of mpClover 24 hours after addition of dox and the absence of GFP“leakiness” in the absence of dox. Note that this population has not be selected on Puro, so it is not a pure population (thus, the negative cells do not contain the donor element.). Moreover, these cells were examined roughly 1 week after a sorting to enrich transfected cells and 2 weeks after transfection so the mpClover populations are stable transfections. Dox inducibility would not function in the absence of integration.
• compositions and methods related to induced pluripotent stem cells iPSCs
• differentiated iPSCs including neural progenitor cells
• vectors used for manipulation of the aforementioned cells methods and compositions related to use of the aforementioned compositions, techniques and composition and use of solutions used therein, and the particular use of the products created through the teachings of the invention.
• Various embodiments of the invention can specifically include or exclude any of these variations or elements.
• the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term“about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon 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 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 invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
• the terms“a” and“an” and“the” and similar references used in the context of describing a particular embodiment of the invention can be construed 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 referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification 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.

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