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Last Updated: January 28, 2022

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Claims for Patent: 6,537,746

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Summary for Patent: 6,537,746
Title: Method for creating polynucleotide and polypeptide sequences
Abstract:The invention provides methods for evolving a polynucleotide toward acquisition of a desired property. Such methods entail incubating a population of parental polynucleotide variants under conditions to generate annealed polynucleotides comprising heteroduplexes. The heteroduplexes are then exposed to a cellular DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants. The resulting polynucleotides are then screened or selected for the desired property.
Inventor(s): Arnold; Frances (Pasadena, CA), Shao; Zhixin (Penzberg, DE), Volkov; Alexander (South Pasadena, CA)
Assignee: Maxygen, Inc. (Redwood City, CA)
Application Number:09/205,448
Patent Litigation and PTAB cases: See patent lawsuits and PTAB cases for patent 6,537,746
Patent Claims:1. A method for evolving a polynucleotide toward acquisition of a desired functional property, comprising (a) incubating a population of parental polynucleotide variants having sufficient diversity that recombination between the parental polynucleotide variants can generate more recombinated-polynucleotides than there are parental polynucleotide variants under conditions to generate annealed polynucleotides comprising heteroduplexes; (b) exposing the heteroduplexes to one or more enzymes of a DNA repair system in vitro to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants; (c) screening or selecting the recombined polynucleotide variants for the desired functional property.

2. The method of claim 1, wherein the DNA repair system comprises cellular extracts.

3. The method of claim 1, wherein the cells are bacterial cells.

4. The method of claim 1 further comprising introducing the products of step (b) into cells.

5. The method of claim 4, wherein the introducing step selects for transformed cells receiving recombinant polynucleotides resulting from resolution of heteroduplexes in step (b) relative to transformed cells receiving polynucleotides resulting from resolution of homoduplexes in step (b).

6. A method for evolving a polynucleotide toward acquisition of a desired functional property, comprising (a) incubating a population of parental polynucleotide variants having sufficient diversity that recombination between the parental polynucleotide variants can generate more recombined polynucleotides than there are parental polynucleotide variants under conditions to generate annealed polynucleotides comprising heteroduplexes; (b) introducing the annealed polynucleotides into cells having a DNA repair system and propagating the cells under conditions to select for cells receiving heteroduplexes relative to cells receiving homoduplexes, and to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants; (c) screening or selecting the recombined polynucleotide variants for the desired functional property.

7. The method of claim 6, wherein the heteroduplexes are exposed to the cellular DNA repair system in vitro.

8. A method for evolving a polynucleotide toward acquisition of a desired functional property, comprising (a) incubating first and second pools of parental polynucleotide variants having sufficient diversity that recombination between the parental polynucleotide variants can generate more recombined polynucleotides than there are parental polynucleotide variants under conditions whereby a strand from any polynucleotide variant in the first pool can anneal with a strand from any polynucleotide in the second pool to generate annealed polynucleotides comprising heteroduplexes; (b) exposing the heteroduplexes to a DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants; (c) screening or selecting the recombined polynucleotide variants for the desired functional property.

9. The method of claim 8, further comprising introducing the heteroduplexes into cells, whereby the heteroduplexes are exposed to the DNA repair system of the cells in vivo.

10. The method of claim 9, wherein the annealed polynucleotides further comprise homoduplexes and the introducing step selects for transformed cells receiving heteroduplexes relative to transformed cells receiving homoduplexes.

11. The method of claim 10, 6, or 5, wherein a first polynucleotide variant is provided as a component of a first vector, and a second polynucleotide variant is provided as a component of a second vector, and the method further comprises converting the first and second vectors to linearized forms in which the first and second polynucleotide variants occur at opposite ends, whereby in the incubating step single-stranded forms of the first linearized vector reanneal with each other to form linear first vector, single-stranded forms of the second linearized vector reanneal with each other to form linear second vector, and single-stranded linearized forms of the first and second vectors anneal with each to form a circular heteroduplex bearing a nick in each strand, and the introducing step selects for transformed cells receiving the circular heteroduplexes or recombinant polynucleotides derived therefrom relative to the linear first and second vector.

12. The method of claim 11, wherein the first and second vectors are converted to linearized forms by PCR.

13. The method of claim 11, wherein the first and second vectors are converted to linearized forms by digestion with first and second restriction enzymes.

14. The method of claim 10, 6 or 5, wherein the population of polynucleotides comprises first and second polynucleotides provided in double stranded form, and the method further comprises incorporating the fist and second polynucleotides as components of first and second vectors, whereby the first and second polynucleotides occupy opposite ends of the first and second vectors, whereby in the incubating step single-stranded forms of the first linearized vector reanneal with each other to form linear first vector, single-stranded forms of the second linearized vector reanneal with each other to form linear second vector, and single-stranded linearized forms of the first and second vectors anneal with each to form a circular heteroduplex bearing a nick in each strand, and the introducing step selects for transformed cells receiving the circular heteroduplexes or recombinant polynucleotides derived therefrom relative to the linear first and second vector.

15. The method of claim 10, 6 or 5, further comprising sealing nicks in the heteroduplexes to form covalently-closed circular heteroduplexes before the introducing step.

16. The method of claim 1, 6 or 8, wherein the population of polynucleotide variants are provided in double stranded form, and the method further comprising converting the double stranded polynucleotides to single stranded polynucleotides before the annealing step.

17. The method of claim 1, 6 or 8 wherein the converting step comprises: conducting asymmetric amplification of the first and second double stranded polynucleotide variants to amplify a first strand of the first polynucleotide variant, and a second strand of the second polynucleotide variant, whereby the first and second strands anneal in the incubating step to form a heteroduplex.

18. The method of claim 17, wherein the first and second double-stranded polynucleotide variants are provided in vector-free form, and the method further comprises incorporating the heteroduplex into a vector.

19. The method of claim 18, wherein the first and second polynucleotides are from chromosomal DNA.

20. The method of claim 1, 6 or 8, further comprising repeating steps (a)-(c) whereby the incubating step in a subsequent cycle is performed on recombinant variants from a previous cycle.

21. The method of claim 1, 6 or 8, wherein the polynucleotide variants encode a polypeptide.

22. The method of claim 1, 6 or 8, wherein the population of polynucleotide variants comprises at least 20 variants.

23. The method of claim 1, 6 or 8, wherein the population of polynucleotide variants are at least 10 kb in length.

24. The method of claim 1, 6 or 8, wherein the population of polynucleotide variants comprises natural variants.

25. The method of claim 1, 6 or 8, wherein the population of polynucleotides comprises variants generated by mutagenic PCR.

26. The method of claim 1, 6 or 8, wherein the population of polynucleotide variants comprises variants generated by site directed mutagenesis.

27. The method of claim 1, 6 or 8, further comprising at least partially demethylating the population of variant polynucleotides.

28. The method of claim 27, whether the at least partially demethylating step is performed by PCR amplification of the population of variant polynucleotides.

29. The method of claim 27, wherein the at least partially demethylating step is performed by amplification of the population of variant polynucleotides in host cells.

30. The method of claim 29, wherein the host cells are defective in a gene encoding a methylase enzyme.

31. The method of claim 27, wherein the population of variant polynucleotides are double stranded polynucleotides and only one strand of each polynucleotide is at least partially demethylated.

32. The method of claim 1, 6 or 8, wherein the population of variant polynucleotide variants comprises at least 5 polynucleotides having at least 90% sequence identity with one another.

33. The method of claim 1, 6 or 8, further comprising isolating a screened recombinant variant.

34. The method of claim 33, further comprising expressing a screened recombinant variant to produce a recombinant protein.

35. The method of claim 34, further comprising formulating the recombinant protein with a carrier to form a pharmaceutical composition.

36. The method of claim 1, 6 or 8, wherein the polynucleotide variants encode enzymes selected from the group consisting of proteases, lipases, amylases, cutinases, cellulases, amylases, oxidases, peroxidases and phytases.

37. The method of claim 1, 6 or 8, wherein the polynucleotide variants encode a polypeptide selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hyperthalnic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO), and prolactin.

38. The method of claim 1, 6 or 8, wherein the polynucleotide variants encode a plurality of enzymes forming a metabolic pathway.

39. The method of claim 1, 6 or 8, wherein the polynucleotide variants are in concatemeric form.

40. The method of claim 39, wherein the functional property is an enzymatic activity.

41. The method of claim 1, 6 or 8, wherein the at least two polynucleotide variants differ at between 0.1-25% of positions.

42. The method of claim 1, 6 or 8, wherein the functional property is an enzymatic activity.

Details for Patent 6,537,746

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Ferring Pharmaceuticals Inc. NOVAREL chorionic gonadotropin For Injection 017016 1974-01-15 ⤷  Sign up for a Free Trial 2017-12-08
Ferring Pharmaceuticals Inc. NOVAREL chorionic gonadotropin For Injection 017016 1984-12-27 ⤷  Sign up for a Free Trial 2017-12-08
Ferring Pharmaceuticals Inc. NOVAREL chorionic gonadotropin For Injection 017016 1985-02-15 ⤷  Sign up for a Free Trial 2017-12-08
Ferring Pharmaceuticals Inc. NOVAREL chorionic gonadotropin For Injection 017016 1990-02-16 ⤷  Sign up for a Free Trial 2017-12-08
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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