Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
  • C12N5/0623—Stem cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/0306—Animal model for genetic diseases
  • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/60—Transcription factors
  • C12N2501/602—Sox-2
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/60—Transcription factors
  • C12N2501/603—Oct-3/4
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/60—Transcription factors
  • C12N2501/604—Klf-4
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/60—Transcription factors
  • C12N2501/606—Transcription factors c-Myc
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/60—Transcription factors
  • C12N2501/608—Lin28
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
  • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• Canavan disease is a devastating neurological disease with symptoms that appear in early infancy and progress rapidly. Possible symptoms include mental retardation, loss of acquired motor skills, feeding difficulties, abnormal muscle tone, unusually large head, paralysis, blindness, and hearing loss.
• Canavan disease is caused by genetic mutations in the aspartoacylase (ASPA) gene, which encodes a metabolic enzyme synthesized by oligodendrocytes in the brain 15 .
• ASPA breaks down N-acetyl-aspartate (NAA), a highly abundant amino acid derivative in the brain. The cycle of production and breakdown of NAA appears to be critical for maintaining the white matter of the brain, which consists of nerve fibers covered by myelin.
• Signs of Canavan disease include lack of ASPA activity, accumulation of NAA in the brain, and spongy degeneration and demyelination of the brain.
• Lithium has been evaluated in Canavan disease animal model and patients and has been shown to reduce NAA level and induce a trend toward normal myelin development in CD-like rats and CD patients. However, it fails to improve the motor function of Canavan patients. Dietary glyceryl triacetate and triheptanoin have been tested in Canavan disease animal models, with improvement in myelination and motor performance observed in treated mice. However, no reduction in NAA levels and only partial amelioration of pathological features were observed. To date, none of these approaches has resulted in complete rescue of the disease phenotypes. There is neither a cure nor a standard treatment for this disease yet.
• this disclosure relates to a method of treating Canavan disease in a subject.
• the method entails restoring ASPA enzymatic activities in the subject by expressing exogenous wild type ASPA gene in the brain of the subject.
• the ASPA enzymatic activities are restored by providing wild type ASPA-expressing neural precursor cells, including neural progenitor cells (NPCs), glial progenitor cells, and oligodendroglial progenitor cells, to the brain of the subject.
• NPCs neural progenitor cells
• glial progenitor cells oligodendroglial progenitor cells
• this disclosure relates to neural precursor cells, including NPCs, glial progenitor cells, and oligodendroglial progenitor cells, which express an exogenous wild type ASPA gene produced by a process comprising the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluhpotent stem cells (iPSCs), introducing wild type ASPA gene in the reprogrammed or converted iPSCs to obtain genetically corrected iPSCs which express wild type ASPA, and differentiating the genetically corrected iPSCs into neural precursor cells, including NPCs, glial progenitor cells and oligodendroglial progenitor cells.
• iPSCs induced pluhpotent stem cells
• the neural precursor cells including NPCs, glial progenitor cells and oligodendroglial progenitor cells, which express an exogenous wild type ASPA gene are produced by a process comprising the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), differentiating the reprogrammed iPSCs into neural precursor cells, and introducing wild type ASPA gene into the neural precursor cells to obtain genetically corrected neural precursor cells which express wild type ASPA.
• iPSCs induced pluripotent stem cells
• this disclosure relates to a method of treating Canavan disease in a subject.
• the method entails the steps of reprogramming or converting somatic cells isolated from the subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), introducing wild type ASPA gene in the reprogrammed or converted iPSCs to obtain genetically corrected iPSCs which express wild type ASPA, differentiating the genetically corrected iPSCs into neural precursor cells, including NPCs, glial progenitor cells and oligodendroglial progenitor cells, and transplanting the neural precursor cells into the brain of the subject.
• iPSCs induced pluripotent stem cells
• the method entails the steps of reprogramming or converting somatic cells isolated from the subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), differentiating the iPSCs into neural precursor cells, including NPCs, glial progenitor cells and oligodendroglial progenitor cells, introducing wild type ASPA gene into the neural precursor cells to obtain genetically corrected neural precursor cells which express wild type ASPA, and transplanting the genetically corrected neural precursor cells into the brain of the subject.
• iPSCs induced pluripotent stem cells
• the somatic cells include but are not limited to fibroblasts, blood cells, urinary cells, adipocytes, keratinocytes, dental pulp cells and other easily accessible somatic cells.
• the somatic cells isolated from the subject suffering from Canavan disease are converted into iPSCs in the presence of one or more reprogramming factors comprising OCT4, SOX2, KLF4, LIN28, p53 shRNA and MYC (such as c-MYC and L-MYC).
• the reprogramming is carried out via episomal reprogramming or viral transduction.
• this disclosure relates to a method of producing wild type ASPA-expressing neural precursor cells which serve as a source of the ASPA enzyme as well as neural precursors to generate WT ASPA-expressing oligodendrocyte progenitor cells (OPCs) and oligo dendrocytes for treating Canavan disease.
• OPCs oligodendrocyte progenitor cells
• the method includes the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), introducing wild type ASPA gene in the reprogrammed or converted iPSCs to obtain genetically corrected iPSCs which express wild type ASPA, and differentiating the genetically corrected iPSCs into neural precursor cells, including NPCs, glial progenitor cells, and oligodendroglial progenitor cells.
• iPSCs induced pluripotent stem cells
• the method includes the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), differentiating the iPSCs into neural precursor cells, including NPCs, glial progenitor cells, and oligodendroglial progenitor cells, and introducing wild type ASPA gene in the precursor cells to obtain genetically corrected precursor cells which express wild type ASPA.
• iPSCs induced pluripotent stem cells
• Figure 1 illustrates characterization of iPSCs derived from WT and CD patient fibroblasts. Expression of human ESC markers in CD iPSCs. CD iPSCs derived from three patients, CD patient 1 , CD patient 2, and CD patient 3, expressed human pluripotency factors OCT4 and NANOG, and the human ESC cell surface marker SSEA4, TRA-1 -60 and TRA-1 -81 . The WT iPSCs derived from IMR90 cells were included as the WT control. Scale bar: 100 ⁇ .
• Figures 2A-2D illustrate that CD patient iPSCs and ASPA iPSCs express human ESC markers.
• Figure 2A shows RT-PCR analysis of endogenous (endo) OCT4, SOX2, and NANOG expression in WT, CD and ASPA iPSCs.
• CD patient fibroblasts (fib) were included as negative controls. Actin was included as a loading control.
• Figure 2B and 2C show RT-PCR analysis of exogenous (exo) reprogramming factors in WT, CD and ASPA iPSCs. Human ESCs were included as a negative control and plasmid DNAs expressing individual factors were included as a positive control.
• Figure 2D shows the karyotype of control and CD iPSCs.
• Figures 3A-3F illustrate characterization of mutations in CD iPSCs and validation of the pluripotency of iPSCs.
• Figure 3A shows that CD iPSCs contained patient-specific ASPA mutations.
• Figure 3B shows validation of iPSC pluripotency in vitro.
• CD1 , CD2 and CD3 iPSCs were able to differentiate into all three germ layers, SOX17-positive endoderm, SMA-positive mesoderm, and TUJ1 -positive ectoderm in EB formation assays.
• WT iPSCs derived from IMR90 cells were included as a control.
• Figure 3C and 3D show validation of iPSC pluripotency in vivo.
• CD1 iPSCs 3C
• CD2 iPSCs and CD3 iPSCs 3D
• Figures 3E & 3F show bisulfite sequencing analysis of OCT4 and NANOG promoter regions in parental CD1 fibroblasts (fib) and CD1 iPSCs. Open and closed circles indicate unmethylated and methylated CpGs, respectively.
• Figures 4A-4M illustrate that the ASPA iPSCs contain the WT ASPA gene and express pluripotency factors.
• Figures 4A, 4D, and 4G show that genomic DNA sequencing confirms the presence of the WT ASPA sequence in ASPA1 , ASPA2, and ASP A3 iPSCs.
• Figures 4B, 4E, and 4H show expression of the human pluripotency factors OCT4 and NANOG in ASPA1 , ASPA2, and ASPA3 iPSCs. Nuclei Dapi staining is shown in blue. Scale bar: 100 ⁇ .
• Figures 4C, 4F, and 4I show expression of human ESC cell surface markers SSEA4, TRA-1 -60 and TRA-1 - 8 in ASPA iPSCs. Nuclei Dapi staining is shown in blue.
• Figure 4J shows that genomic DNA sequencing confirms the presence of WT ASPA sequence in ASPA1 , ASPA2 iPSCs.
• Figures 4K and 4L show expression of transduced ASPA in the ASPA1 iPSCs as revealed by RT-PCR (4K) and Western blot analyses (4L). GAPDH and Tubulin were included as loading controls.
• Figure 4M shows validation of the developmental potential of the ASPA1 iPSCs, ASPA2 iPSCs and ASP A3 iPSCs in vivo.
• ASPA1 iPSCs After injection into immunodeficient NSG mice, the ASPA1 iPSCs, ASPA2 iPSCs and ASPA3 iPSCs were able to form teratomas containing tissues characteristic of each of the three germ layers. Scale bar: 100 ⁇ .
• Figures 5A-5G illustrate characterization of the ASPA1 NPCs.
• Figure 5A shows immunostaining for NPC markers PAX6, SOX2, NCAD, SOX1 , and NESTIN in NPCs derived from the WT, CD1 , and ASPA1 iPSCs. Nuclei Dapi staining is shown in blue. Scale bar: 50 ⁇ .
• Figure 5B shows expression of ASPA and NPC markers in the ASPA1 NPCs as revealed by RT-PCR. The WT and CD1 NPCs were included as controls. GAPDH was included as a loading control.
• Figure 5C shows lack of expression of pluripotency factors in the WT, CD1 , and ASPA1 NPCs as revealed by RT-PCR.
• Figures 5E and 5F show immunostaining pre-OPCs derived from CD1 NPCs or ASPA1 NPCs using OLIG2 and NKX2.2 (5E) or live staining of OPCs derived from CD1 NPCs or ASPA1 NPCs using 04 (5F). Scale bar: 100 ⁇ for 5E and 50 ⁇ for 5F.
• Figure 5G shows FACS analysis of CD1 NPCs and ASPA1 NPCs.
• Figures 6A and 6B illustrate that the ASPA1 NPCs survived and expressed OLIG2 in transplanted CD mouse brains.
• Figure 6A shows that the ASPA1 NPCs were transplanted into neonatal CD mice. One month after transplantation, the mouse brains were harvested and immunostained with antibodies for human nuclear antigen and OLIG2. Scale bar: 100 ⁇ .
• FIGs 7A-7C illustrate that the ASPA1 NPCs gave rise to OLIG2+ cells in transplanted CD mouse brains.
• Figure 7A shows that the ASPA1 NPCs were transplanted into neonatal CD mice. Three months after transplantation, the mouse brains were harvested and immunostained with antibodies for human nuclear antigen (green) and OLIG2 (red).
• Figure 7B shows that three months after transplantation, the mouse brains were harvested and immunostained with antibodies for human nuclear antigen (green) and MBP (red). The enlarged images of the human nuclear antigen (green) and MBP (red)-double positive cells pointed by the arrows were shown in the lower panels.
• Figure 7C shows that the engrafted human cells differentiated into GFAP+ (red) glial cells.
• Scale bar 100 ⁇ for panel A, 63 ⁇ for the upper panels and 10 ⁇ for the lower panels of panel B, and 63 ⁇ for panel C.
• Figures 8A-8E illustrate that the ASPA1 NPCs reduced NAA level and vacuolation in CD mice.
• Figure 8D and 8E show reduced vacuolation in ASPA1 NPC- transplanted CD mouse brains.
• H&E staining of thalamus, cerebellum and brain stem in control CD mice and ASPA1 N PC-transplanted CD mice is shown in panel 8D and quantification of percent vacuolation is shown in panel 8E.
• FIGS 9A-9E illustrate that the ASPA1 NPCs improved myelination and motor function in CD mice.
• Figures 9A and 9B show rescued myelination in ASPA1 NPC-transplanted CD mice. Intact and thick myelin sheaths were detected in brains of 3-month-old wild type (WT) mice, whereas splitting and thinner myelin sheaths were seen in brains of littermate CD mice. Myelin sheaths in CD mice transplanted with the ASPA1 NPCs for three months appeared more intact and thicker. Images of brain stem region are shown. Scale bar: 1 ⁇ . Arrows point to myelin sheaths.
• Figure 9C shows transplantation of the ASPA1 NPCs rescued the weight loss in CD mice.
• Figures 10A-10D illustrate that the ASPA2 NPCs and ASP A3 NPCs exhibited potent ASPA enzymatic activity in vitro.
• Figure 10A shows expression of the NPC markers NESTIN and SOX1 in the ASPA2 NPCs and ASP A3 NPCs by immunostaining. Nuclei Dapi staining is shown in blue in the merged images. Scale bar: 100 ⁇ .
• Figure 10B shows lack of expression of the pluripotency factors OCT4 and NANOG in the ASPA2 NPCs and ASP A3 NPCs as revealed by RT-PCR. ESCs and CD2, CD3 fibroblasts (Fib) were included as positive and negative controls, respectively.
• Figure 10C shows FACS analysis of the ASPA2 NPCs and ASP A3 NPCs.
• Figures 1 1 A-1 1 C demonstrate that the Aspa nur7/nur7 /Rag2 "/" (Aspa nur7 /Rag2 "/” ) mice exhibited similar pathological features to the parental Aspa nur7/nur7 (Aspa nur7 ) mice.
• Figures 1 1 B and 1 1 C show similar vacuolation in the brains of Aspa nur7 /Rag2 "/” mice and the parental Aspa nur7 mice.
• FIG. 12A-12B demonstrate that the ASPA1 NPCs gave rise to OLIG2+ oligodendroglial lineage cells and MBP+ oligodendrocytes in CD mouse brains.
• Figure 12A shows quantification of the percentage of human nuclear antigen (hNu)+and OLIG2+ cells from total grafted human cells in the ASPA1 NPC- transplanted CD brains three months after engraftment.
• FIG. 12B is an orthogonal view to show co-staining for human nuclear antigen and MBP in the ASPA1 NPC-transplanted CD mouse brains. Scale bar: 10 ⁇ .
• FIG. 13 shows that the ASPA1 NPCs can differentiate into GFAP+ cells in transplanted CD mouse brains.
• the ASPA1 NPCs were transplanted into neonatal CD mice. Three months after transplantation, the mouse brains were harvested and immunostained with antibodies for human nuclear antigen (green) and GFAP (red). Scale bar: 63 ⁇ .
• Figures 14A and 14B show that the ASPA2 and ASP A3 NPCs survived and expressed OLIG2 and GFAP in transplanted CD mouse brains.
• the ASPA2 NPCs and ASPA3 NPCs were transplanted into neonatal CD mice.
• Figure 14A shows that three months after transplantation, the mouse brains were harvested and immunostained with antibodies for human nuclear antigen (green) and OLIG2 (red). Scale bar: 25 ⁇ .
• Figure 14B shows that three months after transplantation, the mouse brains were harvested and immunostained with antibodies for human nuclear antigen (green) and GFAP (red). Scale bar: 50 ⁇ .
• Figures 15A-15G show that the ASPA2 and ASPA3 NPCs reduced vacuolation and improved motor function in CD mice.
• Figure 15A shows that CD mice transplanted with the ASPA2 NPCs or ASPA3 NPCs were immunostained for human nuclear antigen (green) and MBP (red). The enlarged images of the human nuclear antigen (green) and MBP (red)-double positive cells pointed by the arrows were shown in the lower panels. Scale bar: 50 ⁇ for the upper panels and 10 ⁇ for the lower panels.
• Figure 15B is an orthogonal view to show co-staining for human nuclear antigen and MBP in the ASPA2 NPC or ASP A3 N PC-transplanted CD mouse brains. Scale bar: 10 ⁇ .
• Figures 16A-16B demonstrate that no tumor formation in the brains of the ASPA1 N PC-transplanted CD mice. Tumor was analyzed through H&E staining 10 months after transplantation with the ASPA1 NPCs. No typical tumor tissue was found in the ASPA1 N PC-transplanted CD mouse brains. Scale bar: 100 ⁇ .
• Figures 17A-17B show low Ki67 index of human engrafted cells at 3 months after transplantation.
• Figure 17A shows that the ASPA-CD1 NPCs were transplanted into CD mouse brains. Three months after transplant, the engrafted brains were immunostained for human nuclear antigen (green) and Ki67 (red). Scale bar: 50 ⁇ .
• iPSCs derived by reprogramming adult human fibroblasts could provide a continuous and autologous donor source for the generation of specific somatic cell types and tissues from individual patients 1"4 .
• Patient-specific iPSCs could provide an unlimited reservoir of disease cell types that otherwise would not be available.
• patient-specific iPSCs are tailored to specific individuals, therefore could reduce the potential for immune rejection.
• recent work demonstrates the feasibility to generate genetically corrected iPSCs from both mice and humans by viral transduction of the wild type (WT) gene or site-specific gene editing. These iPSCs could provide exciting prospects for cell therapy and for studying disease mechanisms.
• WT wild type
• Demyelinating diseases stand out as a particularly promising target for cell-based therapy of central nervous system disorders because remyelination can be achieved with a single cell type, and transplanted myelinogenic cells do not need to integrate into complex neuronal networks 7 .
• myelinogenic potential of rodent and human pluripotent stem cell derivatives have been well documented in various animal models 8"14 .
• the widespread myelination that can be observed in animal models supports the idea that cell therapy provides a potential therapeutic approach in dysmyelinating and demeylinating diseases.
• iPSC-based cell therapy approach is combined with gene therapy approach to generate genetically-corrected patient iPSCs that express the wild type ASPA gene (ASPA iPSCs).
• ASPA iPSCs are differentiated into neural precursor cells, including NPCs, glial progenitor cells, oligodendroglial progenitor cells, and the therapeutic potential thereof is assessed in an immune-deficient Canavan disease mouse model.
• a method of treating Canavan disease in a subject combines patient-specific iPSCs with gene therapy to develop genetically-corrected patient iPSCs that express the WT ASPA gene.
• the ASPA iPSCs were differentiated into NPCs.
• genetic correction can occur at the neural precursor cells level, that is, reprogrammed iPSCs derived from a patient are differentiated into neural precursor cells , and then the wild type ASPA gene is introduced into the neural precursor cells to generate genetically-corrected neural precursor cells.
• the ability of these neural precursors to alleviate the disease phenotypes of CD was tested in a CD mouse model, as demonstrated in the working examples.
• the preclinical efficacy for neural precursor cells derived from genetically corrected patient iPSCs to serve as a therapeutic candidate for CD is demonstrated in the working examples.
• CD is caused by genetic mutation in the ASPA gene
• the CD patient iPSCs or neural precursor cells are genetically corrected by introducing the WT ASPA gene through lentiviral transduction.
• the resultant ASPA iPSCs are differentiated into neural precursor cells.
• the ASPA neural precursor cells exhibit potent ASPA activity, in contrast to CD iPSC-derived CD NPCs that exhibit almost no detectable ASPA activity.
• the ASPA neural precursor cells are transplanted into a CD mouse model that exhibits key pathological phenotypes of CD, including loss of ASPA activity, elevated NAA levels, and extensive sponge degeneration in various brain regions.
• the transplanted ASPA neural precursor cells are able to survive after transplantation and differentiate into oligodendrogial lineage cells.
• transplanted cells are able to exhibit potent ASPA enzymatic activity and reduce NAA levels and sponge degeneration in the brains of transplanted CD mice.
• Transplantation of the ASPA-CD neural precursor cells also can rescue weight loss and behavioral defects of CD mice.
• no tumorigenesis or other adverse effect is observed in the transplanted mice.
• iPSC-derived neural precursor cells can be a cell therapy candidate for CD because neural precursor cells have the capacity to differentiate into oligodendroglial lineage cells that are lost in CD.
• neural precursor cells derived from the genetically corrected, WT ASPA-expressing CD iPSCs can be used because the ASPA-CD neural precursor cells can not only replace the lost oligodendroglial lineage cells but also reconstitute the missing ASPA enzyme.
• iPSCs can provide an unlimited source of cells that are otherwise not possible to obtain for cell replacement therapy due to their easy accessibility and extensive expandability.
• patient-specific iPSCs can provide a source of autologous cells that may avoid immunogenicity associated with allogeneic cell transplantation, therefore offering an option for the treatment of human diseases using cell replacement therapy.
• the differentiated product of iPSCs has not been shown to form teratomas. To address the safety concern associated with potential development of teratoma, it is important to make sure the final iPSC-derived product does not include the undifferentiated cells.
• the method disclosed herein differentiates human iPSCs into neural precursor cells in very high efficiency. As demonstrated in the working examples, FACS analysis of the ASPA1 NPCs showed that more than 98% cells are positive for CD133, a cell surface marker for human NPCs. In contrast, only 0.054% cells are positive for SSEA4, a human ESC surface marker. For ASPA2 NPCs and ASP A3 NPCs, high percentage of CD133-positive cells was also detected. Moreover, cells that were sorted through both positive selection for CD133 and negative election for SSEA4 for ASPA2 and ASP A3 NPC transplantation were used to ensure no contamination of pluripotent stem cells.
• CD has a deficiency in both brain ASPA enzyme and oligodendrocytes
• an ideal strategy for the treatment of CD would be to restore brain ASPA activity and replace dying oligodendrocytes using combined cell therapy and gene therapy.
• the restored ASPA activity could in turn reduce NAA level.
• Transplantation of NPCs expressing the WT human ASPA gene and possessing the ability to differentiate into OPCs and oligodendrocytes offers an appealing therapeutic approach for CD by reconstituting both the missing ASPA enzyme and the lost oligodendroglial lineage cells.
• mouse neural precursor cells transduced with the WT ASPA gene were able to survive and differentiate into oligodendrocytes, and exhibit detectable ASPA activity after transplanting into the brains of CD mice 41 , suggesting that neural precursor cells can be used as a potential source of cell therapy for CD.
• this previous study used mouse cells, which are not clinically applicable 41 .
• increased ASPA activity was only detected at 3 weeks after transplantation, the ASPA activity became undetectable at 5 weeks after transplantation, presumably because of the short term in vivo gene expression from a retroviral vector 41 .
• the effect of the transplanted mouse NPCs on the pathological phenotypes of CD was not studied in the previous study 41 .
• CD patient iPSCs are combined with gene therapy approach to generate genetically-corrected human ASPA iPSCs by transducing CD iPSCs with Ientivirus expressing the WT ASPA gene. These iPSCs are subsequently differentiated into ASPA neural precursor cells. Alternatively, the CD patient iPSCs are differentiated into neural precursor cells and then the WT ASPA gene is introduced into neural precursor cells. The resultant ASPA neural precursor cells served as a source of the ASPA enzyme and as neural precursors to generate WT ASPA-expressing oligodendrocyte progenitor cells (OPCs) and oligodendrocytes.
• OPCs oligodendrocyte progenitor cells
• this disclosure relates to a method of treating Canavan disease in a subject.
• the method entails restoring ASPA enzymatic activities in the subject by expressing exogenous wild type ASPA gene in the brain of the subject.
• the ASPA enzymatic activities are restored by transplanting ASPA neural precursor cells in the brain of the subject.
• These ASPA neural precursor cells serve as a source of the ASPA enzyme as well as neural precursors to generate WT ASPA-expressing oligodendrocyte progenitor cells (OPCs) and oligo dendrocytes.
• ASPA neural precursor cells can be derived from patient-specific iPSCs.
• the method further includes the steps of reprogramming or converting somatic cells isolated from the subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), introducing wild type ASPA gene in the reprogrammed or converted iPSCs to obtain genetically corrected iPSCs which express wild type ASPA, and differentiating the genetically corrected iPSCs into neural precursor cells, including NPCs, glial progenitor cells and oligodendroglial progenitor cells.
• iPSCs induced pluripotent stem cells
• the method further includes the steps of reprogramming or converting somatic cells isolated from the subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), differentiating the iPSCs into neural precursor cells, including NPCs, glial progenitor cells and oligodendroglial progenitor cells, and introducing wild type ASPA gene in the neural precursor cells to obtain genetically corrected neural precursor cells which express wild type ASPA.
• iPSCs induced pluripotent stem cells
• the somatic cells include but are not limited to fibroblasts, blood cells, urinary cells, adipocytes, keratinocytes, dental pulp cells, and other easily accessible somatic cells.
• the somatic cells isolated from the subject suffering from Canavan disease are converted into iPSCs in the presence of one or more reprogramming factors comprising OCT4, SOX2, KLF4, LIN28, p53 shRNA and MYC (such as c-MYC and L-MYC).
• the reprogramming is carried out via episomal reprogramming or viral transduction.
• the wild type ASPA gene is introduced into the reprogrammed iPSCs by transducing the reprogrammed iPSCs with a vector comprising the exogenous wild type ASPA gene. It is within the purview of one of ordinary skill in the art to select a suitable vector and promoter to express the wild type ASPA gene after transduction. In some embodiments, the wild type ASPA gene is introduced by gene editing technology (such as the CRISPR/Cas9 technology).
• this disclosure relates to a method of treating Canavan disease in a subject.
• the method entails the steps of reprogramming or converting somatic cells isolated from the subject suffering from Canavan disease into induced pluhpotent stem cells (iPSCs), introducing wild type ASPA gene in the reprogrammed or converted iPSCs to obtain genetically corrected iPSCs which express wild type ASPA, differentiating the genetically corrected iPSCs into neural precursor cells, and transplanting the neural precursor cells into the brain of the subject.
• iPSCs induced pluhpotent stem cells
• the method entails the steps of reprogramming or converting somatic cells isolated from the subject suffering from Canavan disease into induced pluhpotent stem cells (iPSCs), differentiating the iPSCs into neural precursor cells, introducing wild type ASPA gene in the neural precursor cells to obtain genetically corrected neural precursor cells which express wild type ASPA, and transplanting the genetically corrected neural precursor cells into the brain of the subject.
• iPSCs induced pluhpotent stem cells
• the somatic cells include but are not limited to fibroblasts, blood cells, urinary cells, adipocytes, keratinocytes, dental pulp cells, and other easily accessible somatic cells.
• the somatic cells isolated from the subject suffering from Canavan disease are converted into iPSCs in the presence of one or more reprogramming factors comprising OCT4, SOX2, KLF4, LIN28, p53 shRNA and MYC (such as c-MYC and L-MYC).
• the reprogramming is carried out via episomal reprogramming or viral transduction. It is within the purview of one skilled in the art to select a reprogramming technique to convert the patient somatic cells into iPSCs.
• the iPSCs converted from patient somatic cells contain one or more mutations in the ASPA protein.
• some patients suffering from Canavan disease carry one or more mutations in the ASPA protein, such as A305E, E285A, or G176E mutation, resulting from a codon change of 914C>A , 854A>C, and 527G>A, respectively.
• Some Canavan disease patients may carry other mutations in different regions of the ASPA protein.
• wild type ASPA gene is introduced into the reprogrammed iPSCs by transducing the reprogrammed iPSCs with a vector comprising the exogenous wild type ASPA gene. It is within the purview of one of ordinary skill in the art to select a suitable vector and promoter to express the wild type ASPA gene after transduction.
• the exogenous wild type ASPA gene can be introduced by transducing the patient iPSCs with a lentivirus comprising the wild type ASPA gene.
• the ASPA gene mutation in Canavan disease patient iPSCs can also be corrected by gene editing technologies, such as the CRISPR/Cas9 technology.
• the genetically corrected iPSCs are differentiated in vitro into neural precursor cells, which also express wild type ASPA. After transplanting these ASPA NPCs into the brain of the subject suffering from Canavan disease, the ASPA neural precursor cells can differentiate in vivo into oligodendroglial lineage cells, thereby treating the disease by restoring normal ASPA enzymatic activities.
• the genetic correction occurs at the neural precursor cells level in a similar fashion.
• the CD patient iPSCs are differentiated into neural precursor cells, and then the wild type ASPA gene is introduced to the neural precursor cells by transduction or gene editing, which techniques are known in the art.
• this disclosure relates to a method of producing ASPA neural precursor cells which serve as a source of the ASPA enzyme as well as neural precursors to generate WT ASPA-expressing oligodendrocyte progenitor cells (OPCs) and oligo dendrocytes for treating Canavan disease.
• the ASPA neural precursor cells are derived from patient-specific iPSCs.
• the method includes the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), introducing wild type ASPA gene in the reprogrammed or converted iPSCs to obtain genetically corrected iPSCs which express wild type ASPA, and differentiating the genetically corrected iPSCs into neural precursor cells.
• the method includes the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), differentiating the iPSCs into neural precursor cells, and introducing wild type ASPA gene in the neural precursor cells to obtain genetically corrected neural precursor cells which express wild type ASPA.
• this disclosure relates to neural precursor cells which express an exogenous wild type ASPA gene produced by a process comprising the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), introducing wild type ASPA gene in the reprogrammed or converted iPSCs to obtain genetically corrected iPSCs which express wild type ASPA, and differentiating the genetically corrected iPSCs into neural precursor cells.
• iPSCs induced pluripotent stem cells
• the process comprises the steps of reprogramming or converting somatic cells isolated from a subject suffering from Canavan disease into induced pluripotent stem cells (iPSCs), differentiating the iPSCs into neural precursor cells, and introducing wild type ASPA gene in the neural precursor cells to obtain genetically corrected neural precursor cells which express wild type ASPA.
• neural precursor cells include NPCs, glial progenitor cells and oligodendroglial progenitor cells.
• the somatic cells include but are not limited to fibroblasts, blood cells, urinary cells, adipocytes, keratinocytes, dental pulp cells, and other easily accessible somatic cells.
• the somatic cells isolated from the subject suffering from Canavan disease are converted into iPSCs in the presence of one or more reprogramming factors comprising OCT4, SOX2, KLF4, LIN28, p53 shRNA and MYC (such as c-MYC and L-MYC).
• the reprogramming is carried out via episomal reprogramming or viral transduction.
• the wild type ASPA gene is introduced into the reprogrammed iPSCs by transducing the reprogrammed iPSCs with a vector comprising the exogenous wild type ASPA gene or by genetic editing technology. It is within the purview of one of ordinary skill in the art to select a suitable vector and promoter to express the wild type ASPA gene after transduction.
• treating refers to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
• treating a condition means that the condition is cured without recurrence.
• the terms "subject” and “patient” are used interchangeably in this disclosure.
• the subject or patient suffers from Canavan disease.
• the subject or patient is a mammal.
• the subject or patient is a human.
• iPSCs generation For episomal iPSC derivation, IMR90 fibroblasts (Coriell, 190-10) and CD patient fibroblasts (Coriell , GM04268) were reprogrammed using episomal vectors expressing Oct4, Sox2, Klf4, L-Myc, Lin28 and p53 shRNA as described 32 . Briefly, 5 x 10 5 fibroblasts were electroporated with 1 .25 ⁇ g of each episomal vector, and this day was referred to as day 0. The transfected cells were cultured in fibroblast medium (MEM with NEAA, 15% non-heat-inactivated fetal bovine serum) and medium was changed every other day.
• MEM fibroblast medium
• iPSC clones were picked around day 20 and expanded in E8 medium.
• IMR90 fibroblasts or CD patient fibroblasts (Coriell, GM00059, GM00060 and GM04268) were seeded onto 6-well plates at 1 x 10 5 cells per well in fibroblast medium.
• iPSCs were transduced with freshly prepared viruses of Oct4, Sox2, Klf4 and cMyc as described 1 , and this day was referred to as day 0.
• a second round of viral transduction was performed on day 1 .
• cells were dissociated and split at 1 to 5.
• cells were switched to E8 medium and medium was changed daily thereafter.
• iPSC clones were picked around day 20 and expanded in E8 medium.
• Embryoid body (EB) formation To form EBs, iPSCs were dissociated into small clusters using 0.05 mM EDTA and transferred to E8 medium in T25 flasks. After culturing for two days in E8 medium, EB spheres were switched to human ESC medium containing DMEM/F12, 20% knockout serum, 1 mM L- glutamine, but without bFGF. After two weeks, EBs were plated onto gelatin-coated 12-well plates and cultured for another 2 weeks before immunostaining analysis.
• iPSCs were dissociated with accutase at 1 to 2 dilution in PBS and re-suspended in ice cold mixture of E8 medium and Matrigel (1 : 1 ) at the density of 1 x 10 7 cells/ml.
• 100 ⁇ of cell suspension (1x10 6 cells) were injected subcutaneously into the dorsal flank of immunodeficient Nod Scid Gamma (NSG) mice. Eight to twelve weeks after injection, teratoma was dissected and fixed in formalin. Fixed tissues were embedded in paraffin, sectioned and stained with hemotoxylin and eosin (H&E).
• ASPA viral preparation and transduction To make ASPA- expressing virus, the human ASPA coding sequence was PCR-amplified using human ASPA cDNA (ATCC, MGC-34517) as the template and the PCR product was cloned into the lentiviral vector pSIN-EF2- pur, which was generated by removing Sox2 from pSIN-EF2-Sox2-pur (Addgene, #16577). To package the ASPA- expressing virus, 15 ⁇ g of pSIN-EF2-hASPA-pur, 5 ⁇ g of VSV-G, 5 ⁇ g of REV and 15 ⁇ g of MDL were transfected into HEK 293T cells using calcium phosphate transfection method.
• NPCs neural progenitor cells
• NPCs were derived from human iPSCs following an established protocol 33 . To start neural induction, human iPSCs were dissociated with 0.5 mM EDTA and passaged onto Matrigel-coated plates at 20% confluency in E8 medium.
• NCM-1 Neural Induction Medium 1
• NCM-1 Neural Induction Medium 1
• 50% Advanced DMEM/F12 Life Technologies, 1 1330-032
• 50% Neurobasal Life Technologies, 21 103-049
• N2 Life Technologies, 17502-048
• B27 Life Technologies, 12587-010
• 2 mM GlutaMAX Life Technologies, 35050-061
• 4 ⁇ CHIR99021 D&C Chemicals, DC9703
• 3 ⁇ SB431542 R&D, 1614
• 2 ⁇ Dorsomorphin Sigma, P5499.
• NIM-1 Neural Induction Medium 2
• NIM-2 Neural Induction Medium 2
• NAM-2 Neural Induction Medium 2
• Neurobasal 1x N2, 1x B27, 2 mM GlutaMAX, 4 ⁇ CHIR99021 , 3 ⁇ SB431542 and LDN-193189 (Stemgent, 04-0074) for another 5 days.
• NPCs were then dissociated with Accutase (Sigma, A6964), and maintained in Neural Stem cell Maintenance Medium (NSMM) containing 50% DMEM/F12, 50% Neurobasal, 1 x N2, 1x B27, 2 mM GlutaMAX, 3 ⁇ CHIR99021 , 2 ⁇ SB431542, 20 ng/ml EGF and 20 ng/ml FGF.
• NPCs were treated with 10 ⁇ ROCK inhibitor when dissociated. NPCs were transplanted into neonatal mice within 14 passages.
• iPSC-derived NPCs Differentiation of iPSC-derived NPCs into oligodendrocytes.
• NPCs were switched from NSMM medium (see above) to Neural Induction Medium 3 (NI M-3) containing DMEM/F12, 1 xN2, IxNEAA, 2 mM GlutaMAX, 25 ⁇ g/mL Insulin, 0.1 ⁇ RA and 1 ⁇ SAG and cultured in NIM-3 medium for 4 days with daily medium change. Cells were then dissociated and resuspended in NIM-3, and cultured in NIM-3 for 8 days.
• NI M-3 Neural Induction Medium 3
• PDGF medium containing DMEM/F12, 1xN2, IxNEAA, 2 mM GlutaMAX, 25 ⁇ g/mL Insulin (Sigma, 19278), 5 ng/mL HGF (R&D Systems, 294-HG-025), 10 ng/mL PDGF-AA (R&D Systems , 221 -AA-050), 10 ng/mL IGF-1 (R&D Systems, 291 -G1 -200), 10 ng/mL NT3 (Millipore, GF031 ), 60 ng/mL T3 (Sigma, T2877), 100 ng/mL Biotin (Sigma, 4639), and 1 ⁇ cAMP (Sigma, D0260), for the next 10 days, with medium change every two days.
• Cells were then attached onto matrigel-coated 6-well plates, and cultured in glial medium containing DMEM/F12, 1xN2, IxNEAA, 2 mM GlutaMAX, 25 ⁇ g/mL Insulin, 10 mM HEPES (Sigma, H4034), 60 ng/mL T3, 100 ng/mL Biotin, 1 ⁇ cAMP and 25 ⁇ g/mL ascorbic acid (Sigma, A4403) for 45 days or longer.
• glial medium containing DMEM/F12, 1xN2, IxNEAA, 2 mM GlutaMAX, 25 ⁇ g/mL Insulin, 10 mM HEPES (Sigma, H4034), 60 ng/mL T3, 100 ng/mL Biotin, 1 ⁇ cAMP and 25 ⁇ g/mL ascorbic acid (Sigma, A4403) for 45 days or longer.
• Neonatal mice (P2-P4) were anesthetized on ice for 4 min and then placed onto a stereotaxic device. Cell suspension was injected into neonatal mouse brains using a Hamiliton syringe with 33 gauge needle. The following coordinates, which were modified from a published study 42 , were used for transplantation. The thalamus: (-0.5, 1 , -2.5), the cerebellum: (-2.0, 0.8, -2.5), and the brain stem (-2.0, 0.8, -3.2). All the coordinates are (A, L, V) with reference to Lambda.
• A stands for anteroposterior from midline
• L stands for lateral from midline
• V stands for ventral from the surface of brain, respectively.
• NPCs within 14 passages were transplanted bilaterally into the thalamus, and the cerebellar white matter and the brain stem at 200,000 cells per site in 2 ⁇ _ and six sites per mouse.
• ASPA enzymatic activity assay The ASPA enzymatic assay was developed based on a published protocol 43 . Forty microliter of cell lysates or brain tissue protein supernatants was added to 10 ⁇ of assay buffer, containing 250 mM Tris-HCI, pH 8.0, 250 mM NaCI, 2.5 mM DTT, 0.25% non-ionic detergent, 5 mM CaCI 2 , 5 mM NAA (Sigma, 00920). The reaction mixture was incubated at 37°C for 90 min, then the reaction was stopped by incubating the tubes in boiling water for 3 min. Reaction blank was created by adding 40 ⁇ H 2 0 instead of protein homogenate.
• the reaction mixture was centrifuged at 13,000 rpm for 10 min to remove the precipitates.
• the supernatant was added into the enzyme assay buffer containing 50 mM Tris-HCI, pH 8.0, 50 mM NaCI, 2 mM a-ketoglutarate, 0.15 mM ⁇ - NADH, 1 mg/ml BSA and 10 units each of malate dehydrogenase and Glutamate- Oxaloacetate Transaminase.
• the reaction was incubated at RT for 20 min.
• the supernatant was transferred to a clear 96-well flat bottom plate and absorbance was measured at 340 nm using a plate reader.
• NAA level measurement Aqueous metabolites were extracted from the thalamus, brainstem and cerebellum of indicated mice using the method of perchloric acid (PCA, Sigma, 244253) as described 44 . Briefly, tissues were rapidly chopped into small pieces and collected into 1 .5 ml Eppendorf tubes. 5 ml/g (wet weight basis) of 6% ice-cold PCA was added into each tubes, followed by vortexing for 30 s and incubating the samples on ice for additional 10 min. The mixture was centrifuged at 12,000 g for 10 min at 4°C.
• PCA perchloric acid
• the PCA supernatants were transferred into new tubes and neutralized with 2 M K 2 C0 3 , and placed on ice with lids open to allow C0 2 to escape. Each sample was vortexed and incubated on ice for 30 min to precipitate the potassium perchlorate salt. The supernatant pH was adjusted to 7.4 ⁇ 0.2, then centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was transferred to Eppendorf tubes and frozen on dry ice. The samples were then subjected to NMR analysis at the NMR Core Facility of City of Hope.
• Electron microscopy EM. Mice were deeply anesthetized with isoflurane, and perfused with 0.9% saline followed by 0.1 M Millonig's buffer containing 4% paraformaldehyde (PFA) and 2.5% glutaraldehyde at 37°C. Brain tissues were dissected and post-fixed in the same fixative overnight. A heavy metal staining protocol developed by Dr. Mark Ellisman's group 45 was followed. Target tissues were cut into -150 ⁇ vibratome sections using a Leica VT 1000S vibratome. The vibratome sections were then fixed overnight in 0.15 M cacodylate buffer, pH 7.4, containing 2.5% glutaraldehyde and 2 mM calcium chloride.
• PFA paraformaldehyde
• tissue sections were washed 5 x 3 minutes in 0.15 M cacodylate buffer, pH7.4, containing 2 mM calcium chloride, and then fixed in 0.15 M cacodylate buffer, pH7.4, containing 1 .5% potassium ferrocyanide, 2% aqueous osmium tetroxide, and 2 mM calcium chloride for 1 hr.
• the samples were then placed in 1 % thiocarbohydrazide (Acros Organics) for 20 min, followed by fixing in 2% osmium tetroxide for 30 min.
• the samples were then placed in 1 % aqueous uranyl acetate solution at 4°C for overnight.
• mice The motor performance of mice was tested by rotarod treadmill (Columbus Instruments) as described 34 .
• RNA preparation and RT-PCR analysis were extracted from cells using the TRIazol reagent (Invitrogen, 15596018). Reverse transcription was performed with 1 ⁇ g of RNA using the Tetro cDNA synthesis kit (Bioline, BIO- 65043). The primers used for PCR are listed in Table 2.
• Immunohistochemistry was performed on paraformaldehyde (PFA)-fixed tissue. Animals were deeply anesthetized and transcardially perfused with ice-cold 0.9 % saline followed by 4 % PFA. Perfused brains were removed and post-fixed in 4 % PFA overnight, then cryoprotected with 30% sucrose. Cryoprotected brains were flash frozen and stored at -80 °C. For immunohistochemistry analysis, brain sections were permeabilized in PBS with PBST for 20 min, and washed 3x5 min in PBST. Sections were incubated with primary antibodies (Table 3) at 4 °C for overnight.
• PFA paraformaldehyde
• ASPA1 NPCs were transplanted into CD mice for up to 10 months. Within the 10- month period, the ASPA1 NPC-transplanted CD mice were monitored monthly. Ten months after transplantation, the ASPA1 NPC-transplanted CD mice were perfused, sectioned and subjected to H&E staining or Ki67 staining.
• the third Canavan disease patient has a homozygous mutation at nucleotide 854 of the ASPA gene (854A>C), resulting in a substitution of glutamic acid by alanine at codon 285 (E285A).
• E285A is the predominant mutation (accounting for over 82% of mutations) among the Ashkenazi Jewish population 28,29 , whereas A305E is the most common mutation (60%) in non-Jewish Canavan disease patients 30 .
• G176E is a new ASPA mutation identified in Canavan disease patients as disclosed herein. Normal human fibroblast cells IMR90 were included as the wild type (WT) control (Table 4). Table 4 Wild Type and CD Cells Used in the Study
• CD iPSCs WT and CD patient iPSCs
• the iPSC lines derived from both normal human fibroblasts and CD patient fibroblasts expressed the key human pluripotency genes, OCT4 and NANOG, and the human embryonic stem cell (ESC)-specific surface markers, SSEA4, TRA-1 -60 and TRA-1 - 81 (Fig. 1 ).
• CD1 and CD patient 2 (CD2) iPSCs contain two heterozygous mutations at nucleotide 527 (527G>A) and nucleotide 914 (914C>A) of the ASPA gene, whereas the CD patient 3 (CD3) iPSCs harbor a homozygous mutation at nucleotide 854 of the ASPA gene (854A>C) (Fig. 3A).
• Embryoid body (EB) formation assay was performed to demonstrate the pluripotent potential of the identified CD iPSC clones.
• Both WT and CD iPSCs could differentiate into characteristic SOX17-positive endodermal cells, smooth muscle actin (SMA)-positive mesodermal cells, and ⁇ tubulin (TUJ1 )- positive ectoderm cells (Fig. 3B).
• SMA smooth muscle actin
• TUJ1 ⁇ tubulin- positive ectoderm cells
• CD patient iPSCs were transduced with lentivirus expressing the human WT ASPA gene under the constitutive human EF1 a promoter.
• the genetically corrected CD patient iPSCs were termed ASPA iPSCs.
• the ASPA1 (or ASPA-CD1 ), ASPA2 (or ASPA-CD2), and ASP A3 (or ASPA-CD3) iPSCs were derived from CD patient 1 , CD patient 2, and CD patient 3 iPSCs, respectively.
• the presence of the WT ASPA gene sequence was confirmed in the ASPA1 , ASPA2, and ASPA-3 iPSCs (Figs 4A, 4D, 4G and 4J).
• ASPA iPSCs continued to express the pluripotency factors OCT4 and NANOG and the human ESC surface markers SSEA4, TRA-1 -60 and TRA-1 -81 (Figs. 4B, 4C, 4E, 4F, 4H, 4I).
• RT-PCR confirmed induction of the endogenous OCT4, SOX2 and NANOG expression in ASPA iPSCs (Fig. 2A).
• the exogenous reprogramming factors, OCT4, SOX2, KLF4, LIN28, and MYC were not detectable in these iPSCs (Figs. 2B and 2C).
• the ASPA iPSCs also maintained their developmental potential. After transplanting into immunodeficient NSG mice, the ASPA1 , ASPA2, and ASP A3 iPSCs were able to develop teratomas that contain all three germ layers (Fig. 4M).
• WT, CD1 , and ASPA1 iPSCs were differentiated into neural progenitor cells (NPCs) following a published protocol 33 .
• NPCs neural progenitor cells
• the NPCs derived from all three iPSC lines expressed typical NPC markers, including PAX6, S0X2, N-cadherin, S0X1 , and NESTIN (Figs. 5A & 5B).
• no expression of the pluripotency factors OCT4 and NANOG was detected in either type of NPCs (Fig. 5C).
• the ASPA1 iPSC-derived NPCs (ASPA1 NPCs) also expressed the ASPA gene (Fig. 5B).
• the ASPA1 NPCs exhibited potent ASPA enzymatic activity, compared to CD1 iPSC-derived NPCs (CD1 NPCs), which exhibited no detectable ASPA activity (Fig. 5D). Further differentiation of the ASPA1 NPCs along the oligodendroglial lineage allowed obtaining OLIG2+NKX2.2+ pre-OPCs by day 13 of differentiation, and 04+ OPCs by day 80 of differentiation (Figs. 5E & 5F). Similar results were obtained from CD1 NPCs (Figs. 5E & 5F). These results demonstrate that the ASPA1 NPCs not only possess potent ASPA enzymatic activity but also have the capacity to differentiate into oligodendroglial lineage cells.
• Fluorescence-activated cell sorting revealed that the vast majority of the CD1 NPCs and ASPA1 NPCs are CD133-positive NPCs, with minimal contamination of undifferentiated iPSCs as revealed by the negligible fraction of SSEA4-positive cells (Fig. 5G). Together, these results demonstrate the identity, purity, and potency of the ASPA1 NPCs.
• Example 5 The ASPA NPCs can survive and provide functional rescue in transplanted CD mice
• the Aspa nur7/nur7 mouse contains a nonsense mutation (Q193X) in the ASPA gene 34 . Because Aspa nur7/nur7 mice exhibit key pathological phenotypes resembling those of CD patients, including loss of ASPA enzymatic activity, elevated NAA levels, and extensive sponge degeneration in various brain regions 34 , it is considered an authentic animal model for CD. Therefore, the Aspa nur7/nur7 mouse provides an excellent platform for testing the therapeutic effects of NPCs derived from the genetically-corrected ASPA iPSCs.
• an immunodeficient ASPA nur7//nur7 mouse model was generated by breeding the ASPA nur7/nur7 mice with immunodeficient Rag2 " mice, which lack mature B and T lymphocytes.
• the resultant ASPA nur7/nur7 /Rag2 "/” mice are largely similar to the parental ASPA mice, both of which exhibited substantially reduced ASPA enzymatic activity in the brain, compared to the WT mice (Fig.1 1 A).
• spongy degeneration as revealed by vacuolation is another characteristic feature of CD patients and mouse models.
• the ASPA1 NPCs differentiated from the ASPA1 iPSCs were transplanted into brains of neonatal CD mice. Two-hundred thousand cells in 2 ⁇ _ volume were injected stereotactically into 6 sites of the brain of neonatal CD mice (see Methods). One month after transplantation, mouse brains were harvested and analyzed by immunostaining for human nuclear antigen to identify the transplanted human cells, and for OLIG2, a marker for oligodendroglial lineage cells. The transplanted ASPA1 NPCs were able to survive and express OLIG2 in examined brain regions, including cerebellum and brain stem (Fig. 6A).
• the CD mice that had received ASPA1 NPCs demonstrated substantially improved rotarod performance, compared to CD mice without transplantation (Fig. 6B). These results indicate that the ASPA-expressing NPCs can survive in brains of CD mice, differentiate into oligodendroglial lineage cells and improve the motor function of CD mice.
• the ASPA1 NPCs were transplanted into brains of neonatal CD mice and the mice were allowed to survive for 3 months. Brains of the transplanted mice were then harvested and analyzed by co-staining for human nuclear antigen and the oligodendrocyte lineage transcription factor OLIG2. The transplanted ASPA NPCs were able to survive three months after transplantation and differentiate into oligodendroglial lineage cells (Fig. 7A).
• ASPA enzymatic activity was determined in CD mouse brains harvested three months after ASPA1 NPC transplantation. Potent ASPA enzymatic activity was detected in various brain regions of the ASPA1 N PC-transplanted mice, including thalamus, cerebellum and brain stem, compared to that in CD mouse brains without transplantation (Fig. 8A).
• ASPA deficiency leads to elevated NAA levels in brains of both CD patients and mouse models 15,34"36 . Consistent with elevated ASPA enzymatic activity, reduced NAA level in the ASPA1 N PC-transplanted CD mouse brains, compared to that in CD1 N PC-transplanted CD mouse brains, was detected (Figs. 8B, 8C). These results indicate that transplantation of the ASPA1 NPCs was able to rescue the deficiency of ASPA enzymatic activity and reduce NAA levels, the major defects in both CD patients and mouse models.
• Extensive spongy degeneration is another key pathological feature of CD patients and mouse models, which is revealed by vacuolation in various brain regions 15,34"36 . Consistent with the observation of elevated ASPA enzymatic activity and reduced NAA levels in the brains of the ASPA1 N PC-transplanted CD mice, substantially reduced extent of vacuolation was detected in various brain regions of the ASPA1 NPC-transplanted CD mice, including thalamus, cerebellum and brain stem (Figs. 8D, 8E).
• vacuolation results from myelin destruction in brains of CD mice 34 . Consistent with extensive vacuolation in the brains of CD mice, substantially reduced thickness of myelin sheaths was observed in the brains of CD mice, compared to that from WT mice (Figs. 9A, 9B). The myelin sheaths in the ASPA1 NPC-transplanted CD brains were much thicker than that of untreated CD brains, but resembled more to that of WT brains (Figs. 9A, 9B). These results further support the therapeutic potential of the ASPA1 NPCs for their ability to ameliorate the pathological phenotypes of CD.
• CD mice transplanted with the WT NPCs, CD1 NPCs, or ASPA1 NPCs were tested in two motor skill behavioral paradigms. Three months after transplantation, CD mice that had received intracerebral injection of various NPCs were examined on the accelerating rotarod treadmill, which is designed for testing motor coordination and balance.
• Both the WT NPCs and the ASPA1 NPCs improved rotarod performance in transplanted CD mice substantially, compared to the CD1 NPCs, whereas no significant difference between the mice treated with the WT NPCs or the ASPA1 NPCs were detected (Fig. 9D).
• a hanging wire approach was used to evaluate paw strength as an indication of neuromuscular function
• Substantial enhancement of the grip strength was detected in a hanging wire test in both the WT NPCs and the ASPA1 N PC-transplanted CD mice, compared to that in the CD1 N PC-transplanted CD mice (Fig. 9E).
• Example 6 The ASPA2 NPCs and ASPA3 NPCs can rescue disease phenotypes in CD mice
• ASPA NPCs derived from CD patient 2 and CD patient 3 iPSCs were tested.
• CD2 iPSCs and CD patient 3 iPSCs were transduced with the ASPA-expressing lentivirus, and then these iPSCs were differentiated into NPCs.
• the resultant WT ASPA-expressing NPCs were termed the ASPA2 NPCs and ASP A3 NPCs, respectively.
• Both the ASPA2 NPCs and ASP A3 NPCs expressed typical NPC markers NESTIN and SOX1 (Fig. 10A).
• no expression of the pluripotency factors OCT4 and NANOG was detected in the ASPA2 NPCs and ASP A3 NPCs (Fig. 10B).
• the ASPA2 NPCs and ASP A3 NPCs were sorted by positive selection using the NPC surface marker CD133 and by negative selection using the human ESC surface marker SSEA4.
• the vast majority of the differentiated cells were CD133-positive and SSEA4-negative (Fig. 10C).
• the CD133-positive and SSEA4- negative cell population was harvested for transplantation experiments.
• ASPA activity in the ASPA2 and ASP A3 NPCs was tested, and it was found that both the ASPA2 NPCs and ASP A3 NPCs exhibited potent ASPA activity compared to CD2 NPCs and CD3 NPCs (Fig. 10D).
• the identity, purity and potency of the ASPA2 NPCs and ASP A3 NPCs before transplantation have been demonstrated.
• the sorted CD133-positive and SSEA4-negative ASPA2 NPCs and ASPA3 NPCs were transplanted into brains of neonatal CD mice, as described above and in Methods. Three months after transplantation, cells that were positive for both human nuclear antigen and OLIG2 in the transplanted CD mouse brains were detected (Fig. 14A). Moreover, the engrafted cells were able to give rise to MBP-positive mature oligodendrocytes (Fig. 15A). The presence of grafted cells that are positive for both human nuclear antigen and MBP in the transplanted brains was confirmed by orthogonal view of the confocal images (Fig. 15B). A portion of the transplanted cells gave rise to GFAP-positive astrocytes in the ASPA2 NPC or ASPA3 NPC-tansplanted CD brains (Fig. 14B).
• ASPA enzymatic activity in the brains of CD mice transplanted with the ASPA2 NPCs or ASP A3 NPCs was examined. Potent ASPA enzymatic activity was detected in multiple brain regions of the transplanted brains, including the thalamus, cerebellum and brain stem, compared to that in CD mouse brains without transplantation (Fig. 15C). Similar to the ASPA1 N PC-transplanted CD mice, markedly reduced vacuolation was detected in the ASPA2 NPC or ASP A3 NPC- transplanted CD mouse brains, including the thalamus, cerebellum and brain stem (Figs. 15D & 15E).
• CD mice received the ASPA2 NPCs or ASP A3 NPCs were tested on the accelerating rotarod treadmill. Both the ASPA2 NPCs and ASPA3 NPCs improved rotarod performance in the transplanted CD mice substantially, compared to CD mice without transplantion (Fig. 15F). Substantial enhancement of the grip strength was also detected in a hanging wire test in the CD mice transplanted with either the ASPA2 NPCs or ASP A3 NPCs, compared to that in CD mice without transplantation (Fig. 15G). These results together indicate that transplantation with either the ASPA2 NPCs or ASPA3 NPCs can improve motor functions in a mouse model of CD substantially. These results provide a proof-of-concept that NPCs derived from genetically corrected CD patient iPSCs have therapeutic potential to ameliorate the pathological phenotypes of CD.
• the ASPA1 NPCs were transplanted into the brains of CD mice for up to 10 months. Within these 10 months, the transplanted mice were monitored monthly and no sign of tumor formation was observed (Table 5). At the end of the 10th month, the transplanted mice were harvested and analyzed by H&E staining for further tumor analysis. No typical tumor tissue was found in these sections (Figs. 16A and 16B). The lack of tumor formation in the ASPA1 NPC-transplanted mice was correlated with a very low mitotic index, as revealed by the low percentage of human nuclear antigen-positive and Ki67-positive cells in the ASPA1 NPC-grafted brains (Figs. 17A and 17B).
• Hacein-Bey-Abina S., Garrigue, A. , Wang, G. P., Soulier, J., Lim, A., Morillon, E., Clappier, E., Caccavelli, L, Delabesse, E., Beldjord, K., Asnafi, V., Maclntyre, E., Dal Cortivo, L , Radford, I., Brousse, N., Sigaux, F. , Moshous, D., Hauer, J., Borkhardt, A., Belohradsky, B. H., Wintergerst, U., Velez, M.
• Human embryonic stem cell- derived oligodendrocyte progenitors remyelinate the brain and rescue behavioral deficits following radiation. Cell Stem Ce// 16, 198-210 (2015). Douvaras, P., Wang, J., Zimmer, M., Hanchuk, S., O'Bara, M. A. , Sadiq, S., Sim, F. J., Goldman, J., and Fossati, V.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Cell Biology (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Developmental Biology & Embryology (AREA)
• General Health & Medical Sciences (AREA)
• Neurology (AREA)
• Neurosurgery (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Medicinal Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Virology (AREA)
• Ophthalmology & Optometry (AREA)
• Immunology (AREA)
• Transplantation (AREA)
• Epidemiology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Enzymes And Modification Thereof (AREA)
• Materials For Medical Uses (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (1)

Family

ID=60784859

Family Applications (1)

Country Status (7)

Cited By (1)

Families Citing this family (1)

Citations (4)

Family Cites Families (2)

• 2017
  • 2017-06-22 CN CN201780051943.9A patent/CN110035764B/zh not_active Expired - Fee Related
  • 2017-06-22 EP EP17816246.7A patent/EP3474876A4/en not_active Withdrawn
  • 2017-06-22 CA CA3028714A patent/CA3028714A1/en active Pending
  • 2017-06-22 US US16/311,995 patent/US20190307808A1/en not_active Abandoned
  • 2017-06-22 AU AU2017281374A patent/AU2017281374A1/en not_active Abandoned
  • 2017-06-22 JP JP2018567237A patent/JP2019518775A/ja active Pending
  • 2017-06-22 WO PCT/US2017/038853 patent/WO2017223373A1/en not_active Ceased
• 2022
  • 2022-12-15 JP JP2022199966A patent/JP2023029376A/ja active Pending
• 2023
  • 2023-10-17 US US18/488,859 patent/US20240197789A1/en active Pending

Patent Citations (4)

Also Published As

Similar Documents

Legal Events