Novel modified nucleic acid sequences and methods for increasing mRNA levels and protein expression in cell systems

• US 20050071890A1
• Filed: 09/24/2004
• Published: 03/31/2005
• Est. Priority Date: 10/20/1997
• Status: Abandoned Application

• Current Assignee: Harry Meade, Li How Chen
• Original Assignee: Harry Meade, Li How Chen
• Inventors: Meade, Harry, Chen, Li How
• Application Number:
  US10/949,975
• Publication Number:
  US 20050071890A1
• Time in Patent Office: Days
• US Class Current: 800/7
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 2039/51 comprising whole cells, viruses or DNA/RNA
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/445 Plasmodium
• CPC Class Codes: C07K 14/4732 Casein in foodstuffs A23J
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides modified recombinant nucleic acid sequences (preferably DNA) and methods for increasing the mRNA levels and protein expression of proteins which are known to be, or are likely to be, difficult to express in cell culture systems, mammalian cell culture systems, or in transgenic animals. The preferred “difficult” protein candidates for expression using the recombinant techniques of the invention are those proteins derived from heterologous cells preferably those of lower organisms such as parasites, bacteria, and virus, having DNA coding sequences comprising high overall AT content or AT rich regions and/or mRNA instability motifs and/or rare codons relative to the recombinant expression system to be used.

Claims

1-26. -26. (Cancelled)

27. A method for producing a parasite protein or fragment thereof in milk of a non-human transgenic mammal, comprising:

providing said non-human transgenic mammal whose genome comprises a modified nucleic acid sequence encoding said parasite protein or fragment thereof operably linked to a promoter which directs expression in a mammary gland, wherein said modified nucleic acid sequence has been modified by replacing one or more AT-containing codons of the nucleic acid sequence of said parasite protein or fragment thereof as it naturally occurs in a parasite with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same parasite protein or fragment thereof as derived from said parasite; and

allowing said non-human transgenic mammal to express said parasite protein or fragment thereof in its milk, to thereby produce said parasite protein or fragment thereof.

28. The method of claim 27, wherein all of the codons of the naturally occurring nucleic acid sequence have been replaced with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same parasite protein or fragment thereof as found in the naturally occurring nucleic acid sequence.

29. The method of claim 27, wherein said parasite protein or fragment thereof is expressed in the milk of said non-human transgenic mammal at a level of at least 0.5 mg/ml.

30. The method of claim 27, wherein said parasite protein or fragment thereof is expressed in the milk of said non-human transgenic mammal at a level which is between 1.0 mg/ml and 2.0 mg/ml.

31. The method of claim 27, wherein said parasite protein or fragment thereof as expressed in said non-human transgenic mammal can be detectably expressed in the milk of said transgenic non-human mammal.

32. The method of claim 27, wherein all non-preferred codons are replaced with a codon or codons preferred by a mammalian cell for the purposes of expression.

33. The parasite protein or fragment thereof as produced by the method of claim 27.

34. The method of claim 27 wherein said parasite protein or fragment thereof is a protein, polypeptide or peptide derived from the Plasmodium falciparum protein MSP-1.

35. The method of claim 27 wherein said mammalian cell for the purposes of expression is a mammary epithelial cell.

36. The method of claim 27 wherein said promoter is selected from a group of promoters consisting of:

a) beta-casein;

b) bovine lactoglobulin;

c) whey acid promoter;

d) alpha-ovalbumin; and

e) caprine casein.

37. The method of claim 27 wherein said non-human transgenic mammal is selected from a group of mammals consisting of:

a) caprine;

b) bovine;

c) porcine;

d) rodent; and

e) ovine.

38. The method of claim 27 wherein said modified nucleic acid sequence is modified to provide for the expression of a modified amino acid sequence such at least one glycosylation site on said parasite protein or protein fragment thereof produced by said non-human transgenic mammal is eliminated.

39. A method for producing a parasite protein or fragment thereof in the milk of a non-human transgenic mammal, comprising:

providing said non-human transgenic mammal whose genome comprises a modified nucleic acid sequence encoding said parasite protein or fragment thereof operably linked to a promoter which directs expression in a mammary gland, wherein said nucleic acid sequence of said parasite protein or fragment thereof has been modified by replacing at least a portion of an AUUUA mRNA instability motif in a coding sequence of said parasite protein or fragment thereof as it naturally occurs in a parasite with a codon or codons preferred by a mammalian cell for the purposes of expression so as to remove said AUUUA mRNA instability motif or prevent said AUUUA mRNA instability motif from destabilizing mRNAs encoding said parasite protein or fragment thereof while encoding an amino acid which is the same as the replaced portion of said AUUUA mRNA instability motif;

allowing said non-human transgenic mammal to express said parasite protein or fragment thereof in its milk, to thereby produce said parasite protein or fragment thereof, and wherein the naturally occurring nucleic acid sequence encoding said parasite protein or fragment thereof contains at least one AUUUA instability motif; and

,

40. The method of claim 39, wherein each of said AUUUA mRNA instability motifs present in the naturally occurring nucleic acid have been replaced by a codon or codons preferred by a mammalian cell for the purposes of expression so as to remove said AUUUA mRNA instability motif or prevent said AUUUA mRNA instability motif from destabilizing mRNAs encoding said parasite protein or fragment thereof.

41. The method of claim 39, wherein said modified nucleic acid sequence further comprises at least one additional codon other than a first codon replaced to lower AT content or a nucleic acid sequence modification made to eliminate said AUUUA mRNA instability motif which has been replaced with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same parasite protein or fragment thereof as found in the naturally occurring nucleic acid sequence.

42. The method of claim 39 wherein said parasite protein or fragment thereof is a protein, polypeptide or peptide derived from the Plasmodium falciparum protein MSP-1.

43. The method of claim 39 wherein said mammalian cell for the purposes of expression is a mammary epithelial cell.

44. The method of claim 39 wherein said promoter is selected from a group of promoters consisting of:

a) beta-casein;

b) bovine lactoglobulin;

c) whey acid promoter;

d) alpha-ovalbumin; and

e) caprine casein.

45. The method of claim 39 wherein said non-human transgenic mammal is selected from a group of mammals consisting of:

a) caprine;

b) bovine;

c) porcine;

d) rodent; and

e) ovine.

46. A method for producing a parasite protein or fragment thereof in the milk of a non-human transgenic mammal, comprising:

providing a non-human transgenic mammal whose genome comprises a modified nucleic acid sequence encoding said parasite protein or fragment thereof operably linked to a promoter which directs expression in a mammary gland, wherein said modified nucleic acid sequence has been modified by;

a) replacing at least a portion of an AUUUA mRNA instability motif in the coding sequence of said parasite protein or fragment thereof as it naturally occurs in a parasite with a codon or codons preferred by a mammalian cell for the purposes of expression so as to remove said AUUUA mRNA instability motif or prevent said AUUUA mRNA instability motif from destabilizing mRNAs encoding said parasite protein or fragment thereof while encoding the same amino acid as the replaced portion of said AUUUA mRNA instability motif;

b) replacing one or more AT-containing codons of said modified nucleic acid sequence as it naturally occurs in said parasite with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same amino acid as the replaced codon;

c) allowing said non-human transgenic mammal to express said parasite protein or fragment thereof in its milk, to thereby produce said parasite protein or fragment thereof and wherein the naturally occurring nucleic acid sequence encoding said parasite protein or fragment thereof contains at least one AUUUA instability motif; and

,

47. The method of claim 46, wherein each of said AUUUA mRNA instability motifs present in the naturally occurring nucleic acid have been replaced by a codon or codons preferred by a mammalian cell for the purposes of expression so as to remove said AUUUA mRNA instability motif or prevent said AUUUA mRNA instability motif from destabilizing mRNAs encoding said parasite protein or fragment thereof.

48. The method of claim 46, wherein said modified nucleic acid sequence further comprises at least one additional codon other than a first codon replaced to lower AT content or a nucleic acid sequence modification made to eliminate said AUUUA mRNA instability motif which has been replaced with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same parasite protein or fragment thereof as found in the naturally occurring nucleic acid sequence.

49. The method of claim 46, wherein all of the codons of the naturally occurring nucleic acid sequence have been replaced with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same parasite protein or fragment thereof as found in the naturally occurring nucleic acid sequence.

50. The method of claim 46, wherein said parasite protein or fragment thereof is expressed in the milk of said non-human transgenic mammal at a level of at least 0.5 mg/ml.

51. The method of claim 46, wherein said parasite protein or fragment thereof is expressed in the milk of said non-human transgenic mammal at a level which is between 1.0 mg/ml and 2.0 mg/ml.

52. The method of claim 46, wherein said parasite protein or fragment thereof as expressed in said non-human transgenic mammal can be detectably expressed in the milk of said transgenic non-human mammal.

53. The method of claim 46, wherein all non-preferred codons are replaced with a codon or codons preferred by a mammalian cell for the purposes of expression.

54. The parasite protein or fragment thereof as produced by the method of claim 46.

55. The method of claim 46 wherein said parasite protein or fragment thereof is a protein, polypeptide or peptide derived from the Plasmodium falciparum protein MSP-1.

56. The method of claim 46 wherein said mammalian cell for the purposes of expression is a mammary epithelial cell.

57. The method of claim 46 wherein said promoter is selected from a group of promoters consisting of:

a) beta-casein;

b) bovine lactoglobulin;

c) whey acid promoter;

d) alpha-ovalbumin; and

e) caprine casein.

58. The method of claim 46 wherein said non-human transgenic mammal is selected from a group of mammals consisting of:

a) caprine;

b) bovine;

c) porcine;

d) rodent; and

e) ovine.

59. The method of claim 46 wherein said modified nucleic acid sequence is modified to provide for the expression of a modified amino acid sequence such at least one glycosylation site on said parasite protein or protein fragment thereof produced by said non-human transgenic mammal is eliminated.

60. A transgenic non-human mammal whose germline comprises a modified nucleic acid sequence encoding a parasite protein or fragment thereof operably linked to a promoter which directs expression in a mammary gland, wherein said modified nucleic acid sequence has been modified by replacing at least a portion of an AUUUA mRNA instability motif in a coding sequence as it naturally occurs in a parasite with a codon or codons preferred by a mammalian cell for the purposes of expression so as to remove said AUUUA mRNA instability motif or prevent said AUUUA mRNA instability motif from destabilizing mRNAs encoding said parasite protein or fragment thereof while encoding the same amino acid as the replaced portion of said AUUUA mRNA instability motif and by replacing one or more AT-containing codons of the nucleic acid sequence of said parasite protein or fragment thereof as it naturally occurs in the parasite with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same amino acid as the replaced codon, wherein said non-human transgenic mammal expresses said parasite protein or fragment thereof in its milk and wherein the naturally occurring nucleic acid sequence encoding said parasite protein or fragment thereof contains at least one AUUUA instability motif;

and,

61. The mammal of claim 60, wherein each of said AUUUA mRNA instability motifs present in the naturally occurring nucleic acid have been replaced by a codon or codons preferred by a mammalian cell for the purposes of expression so as to remove said AUUUA mRNA instability motif or prevent said AUUUA mRNA instability motif from destabilizing mRNAs encoding said parasite protein or fragment thereof.

62. The mammal of claim 60, wherein said modified nucleic acid sequence further comprises at least one additional codon other than a first codon replaced to lower AT content or a nucleic acid sequence modification made to eliminate said AUUUA mRNA instability motif which has been replaced with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same parasite protein or fragment thereof as found in the naturally occurring nucleic acid sequence.

63. The mammal of claim 60, wherein all of the codons of the naturally occurring nucleic acid sequence have been replaced with a codon or codons preferred by a mammalian cell for the purposes of expression and encoding the same parasite protein or fragment thereof as found in the naturally occurring nucleic acid sequence.

64. The mammal of claim 60, wherein said parasite protein or fragment thereof is expressed in the milk of said non-human transgenic mammal at a level of at least 0.5 mg/ml.

65. The mammal of claim 60, wherein said parasite protein or fragment thereof is expressed in the milk of said non-human transgenic mammal at a level which is between 1.0 mg/ml and 2.0 mg/ml.

66. The mammal of claim 60, wherein said parasite protein or fragment thereof as expressed in said non-human transgenic mammal can be detectably expressed in the milk of said transgenic non-human mammal.

67. The mammal of claim 60, wherein all non-preferred codons are replaced with a codon or codons preferred by a mammalian cell for the purposes of expression.

68. The parasite protein or fragment thereof as produced by the mammal of claim 60.

69. The mammal of claim 60 wherein said parasite protein or fragment thereof is a protein, polypeptide or peptide derived from the Plasmodium falciparum protein MSP-1.

70. The mammal of claim 60 wherein said mammalian cell for the purposes of expression is a mammary epithelial cell.

71. The mammal of claim 60 wherein said promoter is selected from a group of promoters consisting of:

a) beta-casein;

b) bovine lactoglobulin;

c) whey acid promoter;

d) alpha-ovalbumin; and

e) caprine casein.

72. The mammal of claim 60 wherein said non-human transgenic mammal is selected from a group of mammals consisting of:

a) caprine;

b) bovine;

c) porcine;

d) rodent; and

e) ovine.

73. The mammal of claim 60 wherein said modified nucleic acid sequence is modified to provide for the expression of a modified amino acid sequence such at least one glycosylation site on said parasite protein or protein fragment thereof produced by said non-human transgenic mammal is eliminated.