Pluripotent cell lines and methods of use thereof

• US 9,464,273 B2
• Filed: 12/06/2013
• Issued: 10/11/2016
• Est. Priority Date: 06/24/2008
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.), Tyler Medical Research LLC
• Original Assignee: Parkinson's Institute, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Reijo Pera, Renee Ann, Langston, J. William, Schule, Birgitt, Palmer, Theodore D., Byers, Blake, Nguyen, Ha Nam, Byrne, James Anthony, Cord, Branden John
• Primary Examiner(s): Long, Scott
• Application Number:
  US14/098,890
• Publication Number:
  US 20140356455A1
• Time in Patent Office: 1,040 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/50 Cell markers; Cell surface determinants
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2740/15032 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 : Screening for pharmacological compounds
• CPC Class Codes: G01N 2333/47 : Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 33/5023 : on expression patterns
• CPC Class Codes: G01N 33/5058 : Neurological cells

Abstract

Methods of generating cell lines with a sequence variation or copy number variation of a gene of interest, methods of use thereof, and cell lines with a sequence variation or copy number variation of a gene of interest are provided.

Claims

1. A method of producing a differentiated dopaminergic cell line, the method comprising:

a) producing induced pluripotent stem cells (iPSCs) from somatic cells harvested from a subject diagnosed with a neurodegenerative disease, wherein the somatic cells carry a genetic variation that is associated with the disease; and

b) differentiating the iPSCs towards a dopaminergic cell type, wherein the dopaminergic cell type is associated with the neurodegenerative disease and displays a phenotype of cellular protein aggregation, thereby resulting in the differentiated dopaminergic cell line.

2. The method of claim 1, wherein the somatic cells are fibroblasts.

3. The method of claim 1, wherein the genetic variation is a copy number variation.

4. The method of claim 3, wherein the genetic variation is a triplication.

5. The method of claim 1, wherein the genetic variation is a deletion, an insertion, a complex multi-state variant, a substitution, a transition, a transversion, or a duplication, of one or more nucleotides in a gene that is associated with the disease.

6. The method of claim 1, wherein the neurodegenerative disease is Parkinson'"'"'s disease, Alzheimer'"'"'s disease, diffuse Lewy body disease or any other Lewy body disorder or synucleinopathy, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, frontotemporal dementia with Parkinsonism chromosome 17, Huntington'"'"'s disease, spinocerebellar ataxias, amytropic lateral sclerosis, or Creutzfeld Jakob disease.

7. The method of claim 1, wherein the neurodegenerative disease is Parkinson'"'"'s disease or Parkinson'"'"'s-like disease.

8. The method of claim 7, wherein the genetic variation is in a gene selected from PARK1 (SNCA or α

-synuclein), PARK2 (Parkin), PARK5 (UCHL1), PARK6 (PINK1), PARK7 (DJ-1), PARK8 (LRRK2), and PARK 11 (GIGFY2).

9. The method of claim 8, wherein the gene is PARK1 (SNCA or α

-synuclein).

10. The method of claim 8, wherein the gene is PARK8 (LRRK2).

11. The method of claim 9 wherein the variation is triplication of the SNCA gene.

12. The method of claim 10, wherein the genetic variation comprises a G2019S mutation.

13. The method of claim 1, wherein the iPSCs are differentiated towards a midbrain dopaminergic cell type.

14. The method of claim 1, wherein the iPSCs are differentiated to adopt the cell type within 20 days.

15. The method of claim 1, wherein the iPSCs are produced without the use of a retrovirus or a lentivirus.

16. The method of claim 1, wherein the iPSCs are produced with a method comprising the use of three factors.

17. The method of claim 16, wherein the three factors are OCT4, SOX2, and KLF4.

18. The method of claim 1, wherein the differentiated dopaminergic cell line further displays a phenotype of cell viability, inclusion bodies, dystrophic neurites, apoptosis, necrosis, oxidative stress, mitochondrial dysfunction, or α

-synuclein aggregation.

19. The method of claim 1, wherein the phenotype changes when the differentiated dopaminergic cell line is in contact with a putative therapeutic agent.

20. The method of claim 1, wherein the dopaminergic cell type is a cell type that is affected in the natural progression of the neurodegenerative disease.

21. A method of screening a putative therapeutic agent for usefulness in the treatment of a neurodegenerative disease, the method comprising:

a) producing a differentiated dopaminergic cell line by the method of claim 1;

b) contacting the dopaminergic cell line with an agent;

c) detecting a response to the agent in the dopaminergic cell line; and

d) determining whether the agent is useful in the treatment based on the detected response.