GENOME EDITING OF SENSORY-RELATED GENES IN ANIMALS

• US 20110016541A1
• Filed: 07/23/2010
• Published: 01/20/2011
• Est. Priority Date: 12/04/2008
• Status: Abandoned Application

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Weinstein, Edward, Cui, Xiaoxia, Simmons, Phil
• Application Number:
  US12/842,719
• Publication Number:
  US 20110016541A1
• Time in Patent Office: Days
• US Class Current: 800/3
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0318 Animal model for neurodegenerative disease, e.g. non- Alzheimer's
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Abstract

The present invention provides genetically modified animals and cells comprising edited chromosomal sequences encoding proteins that are associated with nociception or taste disorders. In particular, the animals or cells are generated using a zinc finger nuclease-mediated editing process. Also provided are methods of using the genetically modified animals or cells disclosed herein to screen agents for toxicity and other effects.

Claims

1. A genetically modified animal comprising at least one edited chromosomal sequence encoding a sensory-related protein.

2. The genetically modified animal of claim 1, wherein the edited chromosomal sequence is inactivated, modified, or comprises an integrated sequence.

3. The genetically modified animal of claim 1, wherein the edited chromosomal sequence is inactivated such no functional sensory-related protein is produced.

4. The genetically modified animal of claim 3, wherein inactivated chromosomal sequence comprises no exogenously introduced sequence.

5. The genetically modified animal of claim 3, further comprising at least one chromosomally integrated sequence encoding a functional sensory-related protein.

6. The genetically modified animal of claim 1, wherein the sensory-related protein is chosen from TRPM5, TRPM7, TRPC1, TRPC5, TRPC6, TRPA1, CNR1, CNR2, POMC, CALCA, CRF, PRKACA, PRKACB, PRKAR1A, PRKAR2A, ERAL1, NR2B, LGALS1, TRPV1, SCN9A, OPRM1, OPRD1, OPRK1, and combinations thereof.

7. The genetically modified animal of claim 1, further comprising a conditional knock-out system for conditional expression of the sensory-related protein.

8. The genetically modified animal of claim 1, wherein the edited chromosomal sequence comprises an integrated reporter sequence.

9. The genetically modified animal of claim 1, wherein the animal is heterozygous or homozygous for the at least one edited chromosomal sequence.

10. The genetically modified animal of claim 1, wherein the animal is an embryo, a juvenile, or an adult.

11. The genetically modified animal of claim 1, wherein the animal is chosen from bovine, canine, equine, feline, ovine, porcine, non-human primate, and rodent.

12. The genetically modified animal of claim 1, wherein the animal is rat.

13. The genetically modified animal of claim 4, wherein the animal is rat and the protein is an ortholog of a human sensory-related protein.

14. A non-human embryo comprising at least one RNA molecule encoding a zinc finger nuclease that recognizes a chromosomal sequence encoding a sensory-related protein, and, optionally, at least one donor polynucleotide comprising a sequence encoding the sensory-related protein or an edited sensory-related protein.

15. The non-human embryo of claim 14, wherein the sensory-related protein is chosen from TRPM5, TRPM7, TRPC1, TRPC5, TRPC6, TRPA1, CNR1, CNR2, POMC, CALCA, CRF, PRKACA, PRKACB, PRKAR1A, PRKAR2A, ERAL1, NR2B, LGALS1, TRPV1, SCN9A, OPRM1, OPRD1, OPRK1, and combinations thereof.

16. The non-human embryo of claim 14, wherein the embryo is chosen from bovine, canine, equine, feline, ovine, porcine, non-human primate, and rodent.

17. The non-human embryo of claim 14, wherein the embryo is rat and the protein is an ortholog of a human sensory-related protein.

18. A genetically modified cell, the cell comprising at least one edited chromosomal sequence encoding a sensory-related protein.

19. The genetically modified cell of claim 18, wherein the edited chromosomal sequence is inactivated, modified, or comprises an integrated sequence.

20. The genetically modified cell of claim 18, wherein the edited chromosomal sequence is inactivated such that no functional sensory-related protein is produced.

21. The genetically modified cell of claim 20, further comprising at least one chromosomally integrated sequence encoding a functional sensory-related protein.

22. The genetically modified cell of claim 18, wherein the sensory-related protein is chosen from TRPM5, TRPM7, TRPC1, TRPC5, TRPC6, TRPA1, CNR1, CNR2, POMC, CALCA, CRF, PRKACA, PRKACB, PRKAR1A, PRKAR2A, ERAL1, NR2B, LGALS1, TRPV1, SCN9A, OPRM1, OPRD1, OPRK1, and combinations thereof.

23. The genetically modified cell of claim 18, wherein the cell is heterozygous or homozygous for the at least one edited chromosomal sequence.

24. The genetically modified cell of claim 18, wherein the cell is of bovine, canine, equine, feline, human, ovine, porcine, non-human primate, or rodent origin.

25. The genetically modified cell of claim 18, wherein the cell is of rat origin and the protein is an ortholog of a human sensory-related protein.

26. A method for assessing an effect of an agent in an animal, the method comprising:

a) administering the agent to a genetically modified animal comprising at least one edited chromosomal sequence encoding a sensory-related protein;

b) obtaining a parameter from the genetically modified animal, wherein the parameter is chosen from any one or more of;

i. rate of elimination of the agent or at least one agent metabolite;

ii. circulatory levels of the agent or the at least one agent metabolite;

iii. bioavailability of the agent or the at least one agent metabolite;

iv. rate of metabolism of the agent or the at least one agent metabolite;

v. rate of clearance of the agent or the at least one agent metabolite;

vi. toxicity of the agent or the at least one agent metabolite;

vii. disposition of the agent or the at least one agent metabolite;

viii. extrahepatic contribution to the rate of metabolism or the rate of clearance of the agent or the at least one agent metabolite; and

ix. ability of the agent to modify an incidence or indication of a sensory disorder in the genetically modified animal, wherein the sensory disorder is chosen from a nociception disorder, a taste disorder, or any combination thereof; and

c) comparing the parameter obtained from the genetically modified animal to the selected obtained from a wild-type animal administered the same agent.

27. The method of claim 26, wherein the agent is a pharmaceutically active ingredient, a drug, a toxin, or a chemical.

28. The method of claim 26, wherein the at least one edited chromosomal sequence is inactivated such that no functional sensory-related protein is produced, and wherein the genetically modified animal further comprises at least one chromosomally integrated sequence encoding a functional ortholog of the sensory-related protein.

29. The method of claim 26, wherein the sensory-related protein is chosen from TRPM5, TRPM7, TRPC1, TRPC5, TRPC6, TRPA1, CNR1, CNR2, POMC, CALCA, CRF, PRKACA, PRKACB, PRKAR1A, PRKAR2A, ERAL1, NR2B, LGALS1, TRPV1, SCN9A, OPRM1, OPRD1, OPRK1, and combinations thereof.

30. The method of claim 26, wherein the animal is a rat of a strain chosen from Dahl Salt-Sensitive, Fischer 344, Lewis, Long Evans Hooded, Sprague-Dawley, and Wistar.

31. A method for assessing an indication of a sensory disorder chosen from a nociception disorder, a taste disorder, and combinations thereof in an animal model comprising a genetically modified animal comprising at least one edited chromosomal sequence encoding a sensory-related protein, the method comprising comparing a selected parameter obtained from the animal model to the selected parameter obtained from a wild-type animal, wherein the selected parameter is chosen from:

a) spontaneous behaviors;

b) performance during behavioral testing;

c) physiological anomalies;

d) abnormalities in tissues or cells;

e) biochemical function; and

f) molecular structures.

32. The method of claim 31, wherein the at least one indication of the sensory disorder occurs spontaneously in the animal model.

33. The method of claim 31, wherein the at least one indication of the sensory disorder is promoted by exposure to an exogenous agent chosen from an nocioception-stimulating agent, a taste-stimulating agent, a sensory-related protein, a sensory-related agonist, and a sensory-related antagonist.

34. A method for assessing at least one side effect of a therapeutic compound comprising administering the therapeutic compound to an animal model, wherein the animal model is chosen from a genetically modified animal and a wild-type animal, wherein the genetically modified animal comprises at least one edited chromosomal sequence encoding a sensory-related protein, and assessing at least one or more behaviors chosen from learning, memory, anxiety, depression, addiction, sensory-motor function, taste preference, and odor preference.

35. The method of claim 34, wherein the therapeutic compound is chosen from a novel therapeutic compound and a novel combination of known therapeutic agents.

36. The method of claim 34, wherein the animal model further comprises a wild-type animal.

37. The method of claim 34, wherein the treatment with the therapeutic compound is self-administered.

38. The method of claim 34, wherein the treatment with the therapeutic compound is administered by incorporating the therapeutic compound in an amount of water, food, or supplemental material provided to the animal model.