Methods and compositions for regulated expression of multiple nucleic acids

• US 20100303776A1
• Filed: 04/16/2010
• Published: 12/02/2010
• Est. Priority Date: 04/16/2009
• Status: Abandoned Application

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Samulski, Richard Jude, Chou, Kyson Xiaohuai
• Application Number:
  US12/799,095
• Publication Number:
  US 20100303776A1
• Time in Patent Office: Days
• US Class Current: 424/93.21
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/51 Specially adapted vectors
• CPC Class Codes: C12N 2799/025 where the vector is derived from a parvovirus
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2840/44 being a specific part of the splice mechanism, e.g. donor, acceptor

Abstract

The present invention provides a cell comprising a first heterologous nucleic acid construct and a second heterologous nucleic acid construct, wherein each of said first and second heterologous nucleic acid constructs comprises: A. a first nucleotide sequence encoding a nucleotide sequence of interest (NOI); and B. a second intronic nucleotide sequence comprising: i) a first set of splice elements defining a first intron that is removed by splicing to produce a first RNA molecule encoded by said first nucleotide sequence in the absence of activity at a second set of splice elements; and ii) the second set of splice elements defining a second intron different from said first intron, wherein said second intron is removed by splicing to produce no RNA molecule and/or to produce a second RNA molecule that is not encoded by said first nucleotide sequence, wherein each said first nucleotide sequence of each of said first and second heterologous nucleic acid constructs is different from one another and wherein each said second intronic nucleotide sequence of each of said first and second heterologous nucleic acid constructs is different from one another.

Claims

1. A cell comprising a first heterologous nucleic acid construct and a second heterologous nucleic acid construct, wherein each of said first and second constructs comprises:

A. a first nucleotide sequence encoding a nucleotide sequence of interest (NOI), wherein the NOI is heterologous to the cell; and

B. a second intronic nucleotide sequence operably associated with said first nucleotide sequence, wherein said second intronic nucleotide sequence is heterologous to said first nucleotide sequence and heterologous to said cell, and further wherein said second intronic nucleotide sequence comprises;

i) a first intron defined by a first set of splice elements that is removed by splicing to produce a first RNA molecule encoded by said first nucleotide sequence, under conditions whereby removal of a second intron defined by a second set of splice elements is prevented; and

ii) said second intron defined by said second set of splice elements, wherein said second intron is different from said first intron, and wherein under conditions whereby removal of said second intron is not prevented and said second intron is removed by splicing, no RNA molecule and/or a second RNA molecule that is not encoded by said first nucleotide sequence is produced,

further wherein said NOI of said first nucleotide sequence of each of said first and second constructs is different from one another and wherein said second intronic nucleotide sequence of each of said first and second heterologous nucleic acid constructs is different from one another.

2. The cell of claim 1, wherein said first and second heterologous nucleic acid constructs are present in said cell as separate nucleic acid constructs.

3. The cell of claim 1, wherein said first and second heterologous nucleic acid constructs are present in said cell as a single nucleic acid construct.

4. The cell of claim 1, wherein said first nucleotide sequence encodes a nucleotide sequence of interest (NOI) selected from the group consisting of:

a) a nucleotide sequence encoding a protein or peptide;

b) a nucleotide sequence encoding a product having activity as an interfering RNA;

c) a nucleotide sequence encoding a product having enzymatic activity as an RNA;

d) a nucleotide sequence encoding a ribozyme;

e) a nucleotide sequence encoding an antisense sequence;

f) a nucleotide sequence encoding a small nuclear RNA (snRNA); and

g) any combination of (a)-(f) above

5. The cell of claim 1, wherein said second intronic nucleotide sequence of at least one of said first or second heterologous nucleic acid constructs is selected from the group consisting of:

a1) the nucleotide sequence of SEQ ID NO;

92 (S0 257 by intron);

b1) the nucleotide sequence of SEQ ID NO;

2 (S0−

GT);

c1) the nucleotide sequence of SEQ ID NO;

1 (S0−

CT);

d1) the nucleotide sequence of SEQ ID NO;

4 (S0−

GT+CT);

e1) the nucleotide sequence of SEQ ID NO;

3 (51);

f1) the nucleotide sequence of SEQ ID NO;

5 (S1+CT);

g1) the nucleotide sequence of SEQ ID NO;

6 (M3);

h1) the nucleotide sequence of SEQ ID NO;

7 (M3+CT);

i1) the nucleotide sequence of SEQ ID NO;

8 (M6);

j1) the nucleotide sequence of SEQ ID NO;

9 (M6+CT);

k1) the nucleotide sequence of SEQ ID NO;

14 (M3+S1);

l1) the nucleotide sequence of SEQ ID NO;

16 (M3+S1+CT);

m1) the nucleotide sequence of SEQ ID NO;

15 (M6+S1);

n1) the nucleotide sequence of SEQ ID NO;

17 (M6+S1+CT);

o1) the nucleotide sequence of SEQ ID NO;

22, which comprises the sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉GTX₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO;

324), wherein X of any of X₁-X₉ and X₁₀-X₁₄ can be A, C, T or G, in any combination;

p1) the nucleotide sequence of SEQ ID NO;

23, which comprises the sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉GTX₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO;

324), wherein X of any of X₁-X₉ and X₁₀-X₁₄ can be A, C, T or G, in any combination;

q1) the nucleotide sequence of SEQ ID NO;

24, which comprises the sequence X₁GX₃GX₅X₆X₇AGGTAATAX₁₄ (SEQ ID NO;

325), wherein X of any of X₁, X₃-X₇ and X₁₄ can be A, C, T or G, in any combination;

r1) the nucleotide sequence of SEQ ID NO;

25, which comprises the sequence X₁GX₃GX₅X₆X₇AGGTAATAX₁₄ (SEQ ID NO;

325), wherein X of any of X₁, X₃-X₇ and X₁₄ can be A, C, T or G, in any combination;

s1) the nucleotide sequence of SEQ ID NO;

26, which comprises the sequence X₁GX₃GX₅X₆X₇AGGTAAGTX₁₄ (SEQ ID NO;

326), wherein X of any of X₁, X₃, X₅-X₇ and X₁₄ can be A, C, T or G, in any combination;

t1) the nucleotide sequence of SEQ ID NO;

27, which comprises the sequence X₁GX₃GX₆X₆X₇AGGTAAGTX₁₄ (SEQ ID NO;

326), wherein X of any of X₁, X₃, X₅-X₇ and X₁₄ can be A, C, T or G, in any combination;

u1) the nucleotide sequence of SEQ ID NO;

28, which comprises the sequence X₁X₂X₃X₄X₆X₆AAGGTAATAG (SEQ ID NO;

327), wherein X of any of X₁,-X₆ can be A, C, T or G, in any combination;

v1) the nucleotide sequence of SEQ ID NO;

29, which comprises the sequence X₁X₂X₃X₄X₆X₆AAGGTAATAG (SEQ ID NO;

327), wherein X of any of X₁,-X₆ can be A, C, T or G, in any combination;

w1) the nucleotide sequence of SEQ ID NO;

30, which comprises the sequence X₁GX₃X₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

328), wherein X of any of X₁ and X₃-X₇ can be A, C, T or G, in any combination;

x1) the nucleotide sequence of SEQ ID NO;

31, which comprises the sequence X₁GX₃X₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

328), wherein X of any of X₁, and X₃-X₇ can be A, C, T or G, in any combination;

y1) the nucleotide sequence of SEQ ID NO;

32, which comprises the sequence X₁X₂GX₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

329), wherein X of any of X₁, X₂ and X₄-X₇ can be A, C, T or G, in any combination;

z1) the nucleotide sequence of SEQ ID NO;

33, which comprises the sequence X₁X₂GX₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

329), wherein X of any of X₁, X₂ and X₄-X₇ can be A, C, T or G, in any combination;

a2) the nucleotide sequence of SEQ ID NO;

34, which comprises the sequence X₁X₂X₃GX₆X₆X₇AGGTAATAG (SEQ ID NO;

330), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

b2) the nucleotide sequence of SEQ ID NO;

35, which comprises the sequence X₁X₂X₃GX₆X₆X₇AGGTAATAG (SEQ ID NO;

330), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

c2) the nucleotide sequence of SEQ ID NO;

36, which comprises the sequence X₁X₂X₃X₄TX₆X₇AGGTAATAG (SEQ ID NO;

331), wherein X of any of X₁-X₄ and X₆-X₇ can be A, C, T or G, in any combination;

d2) the nucleotide sequence of SEQ ID NO;

37, which comprises the sequence X₁X₂X₃X₄TX₆X₇AGGTAATAG (SEQ ID NO;

331), wherein X of any of X₁-X₄ and X₆-X₇ can be A, C, T or G, in any combination;

e2) the nucleotide sequence of SEQ ID NO;

38, which comprises the sequence X_IGGX₄X₆X₆X₇X₈GGTAATAG (SEQ ID NO;

332), wherein X of any of X₁ and X₄-X₈ can be A, C, T or G, in any combination;

f2) the nucleotide sequence of SEQ ID NO;

39, which comprises the sequence X₁GGX₄X₅X₈X₇X₈GGTAATAG (SEQ ID NO;

332), wherein X of any of X₁ and X₄-X₈ can be A, C, T or G, in any combination;

g2) the nucleotide sequence of SEQ ID NO;

40, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAATAG (SEQ ID NO;

333), wherein X of any of X₁, X₂ and X₅-X₆ can be A, C, T or G, in any combination;

h2) the nucleotide sequence of SEQ ID NO;

41, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAATAG (SEQ ID NO;

333), wherein X of any of X₁, X₂ and X₅-X₈ can be A, C, T or G, in any combination;

i2) the nucleotide sequence of SEQ ID NO;

42, which comprises the sequence X₁X₂X₃GTX₈X₇X₈GGTAATAG (SEQ ID NO;

334), wherein X of any of X₁-X₃ and X₆-X₈ can be A, C, T or G, in any combination;

j2) the nucleotide sequence of SEQ ID NO;

43, which comprises the sequence X₁X₂X₃GTX₈X₇X₈GGTAATAG (SEQ ID NO;

334), wherein X of any of X₁-X₃ and X₆-X₈ can be A, C, T or G, in any combination;

k2) the nucleotide sequence of SEQ ID NO;

44, which comprises the sequence X₁X₂X₃X₄TTX₇X₈GGTAATAG (SEQ ID NO;

335), wherein X of any of X₁-X₄ and X₇-X₈ can be A, C, T or G, in any combination;

l2) the nucleotide sequence of SEQ ID NO;

45, which comprises the sequence X₁X₂X₃X₄TTX₇X₈GGTAATAG (SEQ ID NO;

335), wherein X of any of X₁-X₄ and X₇-X₈ can be A, C, T or G, in any combination;

m2) the nucleotide sequence of SEQ ID NO;

46, which comprises the sequence X₁X₂X₃X₄X₅TAX₈GGTAATAG (SEQ ID NO;

336), wherein X of any of X₁-X₅ and X₈ can be A, C, T or G, in any combination;

n2) the nucleotide sequence of SEQ ID NO;

47, which comprises the sequence X₁X₂X₃X₄X₅TAX₈GGTAATAG (SEQ ID NO;

336), wherein X of any of X₁-X₅ and X₈ can be A, C, T or G, in any combination;

o2) the nucleotide sequence of SEQ ID NO;

48, which comprises the sequence X₁X₂X₃X₄X₅X₈AAGGTAAGTG (SEQ ID NO;

337), wherein X of any of X₁-X₆ can be A, C, T or G, in any combination;

p2) the nucleotide sequence of SEQ ID NO;

49, which comprises the sequence X₁X₂X₃X₄X₅X₈AAGGTAAGTG (SEQ ID NO;

337), wherein X of any of X₁-X₆ can be A, C, T or G, in any combination;

q2) the nucleotide sequence of SEQ ID NO;

50, which comprises the sequence X₁GX₃X₄X₅X₆X₇AGGTAAGTG (SEQ ID NO;

338), wherein X of any of X₁ and X₃-X₇ can be A, C, T or G, in any combination;

r2) the nucleotide sequence of SEQ ID NO;

51, which comprises the sequence X₁GX₃X₄X₅X₈X₇AGGTAAGTG (SEQ ID NO;

338), wherein X of any of X₁ and X₃-X₇ can be A, C, T or G, in any combination;

s2) the nucleotide sequence of SEQ ID NO;

52, in any combination, which comprises the sequence X₁X₂GX₄X₅X₈X₇AGGTAAGTG (SEQ ID NO;

339), wherein X of any of X₁-X₂ and X₄-X₇ can be A, C, T or G;

t2) the nucleotide sequence of SEQ ID NO;

53, which comprises the sequence X₁X₂GX₄X₅X₈X₇AGGTAAGTG (SEQ ID NO;

339), wherein X of any of X₁-X₂ and X₄-X₇ can be A, C, T or G, in any combination;

u2) the nucleotide sequence of SEQ ID NO;

54, which comprises the sequence X₁X₂X₃GX₅X₆X₇AGGTAAGTG (SEQ ID NO;

340), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

v2) the nucleotide sequence of SEQ ID NO;

55, which comprises the sequence X₁X₂X₃GX₅X₆X₇AGGTAAGTG (SEQ ID NO;

340), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

w2) the nucleotide sequence of SEQ ID NO;

56, which comprises the sequence X₁X₂X₃X₄TX₆X₇AGGTAAGTG (SEQ ID NO;

341), wherein X of any of X₁-X₄ and X₆-X₇ can be A, C, T or G, in any combination;

x2) the nucleotide sequence of SEQ ID NO;

57, which comprises the sequence X₁X₂X₃X₄TX₈X₇AGGTAAGTG (SEQ ID NO;

341), wherein X of any of X₁-X₁and X₆-X₇ can be A, C, T or G, in any combination;

y2) the nucleotide sequence of SEQ ID NO;

58, which comprises the sequence X₁GGX₄X₅X₆X₇X₈GGTAAGTG (SEQ ID NO;

342), wherein X of any of X₁ and X₄-X₈ can be A, C, T or G, in any combination;

z2) the nucleotide sequence of SEQ ID NO;

59, which comprises the sequence X₁GGX₄X₅X₆X₇X₈GGTAAGTG (SEQ ID NO;

342), wherein X of any of X₁ and X₄-X₈ can be A, C, T or G, in any combination;

a3) the nucleotide sequence of SEQ ID NO;

60, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAAGTG (SEQ ID NO;

343), wherein X of any of X₁-X₂ and X₅-X₈ can be A, C, T or G, in any combination;

b3) the nucleotide sequence of SEQ ID NO;

61, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAAGTG (SEQ ID NO;

343), wherein X of any of X₁-X₂ and X₅-X₈ can be A, C, T or G, in any combination;

c3) the nucleotide sequence of SEQ ID NO;

62, which comprises the sequence X₁X₂X₃GTX₈X₇X₈GGTAAGTG (SEQ ID NO;

344), wherein X of any of X₁-X₂ and X₅-X₈ can be A, C, T or G, in any combination;

d3) the nucleotide sequence of SEQ ID NO;

63, which comprises the sequence X₁X₂X₃GTX₆X₇X₈GGTAAGTG (SEQ ID NO;

344), wherein X of any of X₁-X₃ and X₆-X₈ can be A, C, T or G, in any combination;

e3) the nucleotide sequence of SEQ ID NO;

64, which comprises the sequence X₁X₂X₃X₄TTX₇X₈GGTAAGTG (SEQ ID NO;

345), wherein X of any of X₁-X₄ and X₇-X₅ can be A, C, T or G, in any combination;

f3) the nucleotide sequence of SEQ ID NO;

65, which comprises the sequence X₁X₂X₃X₄TTX₇X₈GGTAAGTG (SEQ ID NO;

345), wherein X of any of X₁-X₄ and X₇-X₈ can be A, C, T or G, in any combination;

g3) the nucleotide sequence of SEQ ID NO;

66, which comprises the sequence X₁X₂X₃X₄X₆TAX₈GGTAATGTG (SEQ ID NO;

346), wherein X of any of X₁-X₅ and X₈ can be A, C, T or G, in any combination;

h3) the nucleotide sequence of SEQ ID NO;

67, which comprises the sequence X₁X₂X₃X₄X₆TAX₈GGTAATGTG (SEQ ID NO;

346), wherein X of any of X₁-X₅ and X₈ can be A, C, T or G, in any combination;

i3) the nucleotide sequence of SEQ ID NO;

68, which comprises the sequence AGCGAATAGGTAATAC (SEQ ID NO;

347);

j3) the nucleotide sequence of SEQ ID NO;

69, which comprises the sequence AGCGAATAGGTAATAC (SEQ ID NO;

347);

k3) the nucleotide sequence of SEQ ID NO;

70, which comprises the sequence CGAGGGCAGGTAATAA (SEQ ID NO;

348);

l3) the nucleotide sequence of SEQ ID NO;

71, which comprises the sequence CGAGGGCAGGTAATAA (SEQ ID NO;

348);

m3) the nucleotide sequence of SEQ ID NO;

72, which comprises the sequence GGTGCCGAGGTAATAT (SEQ ID NO;

349);

n3) the nucleotide sequence of SEQ ID NO;

73, which comprises the sequence GGTGCCGAGGTAATAT (SEQ ID NO;

349);

o3) the nucleotide sequence of SEQ ID NO;

74, which comprises the sequence GGTGACTAGGTAATAC (SEQ ID NO;

350);

p3) the nucleotide sequence of SEQ ID NO;

75, which comprises the sequence GGTGACTAGGTAATAC (SEQ ID NO;

350);

q3) the nucleotide sequence of SEQ ID NO;

76, which comprises the sequence CGAGGGCAGGTAATAT (SEQ ID NO;

351);

r3) the nucleotide sequence of SEQ ID NO;

77, which comprises the sequence CGAGGGCAGGTAATAT (SEQ ID NO;

351);

s3) the nucleotide sequence of SEQ ID NO;

78, which comprises the sequence AGCGCAGAGGTAATAA (SEQ ID NO;

352);

t3) the nucleotide sequence of SEQ ID NO;

79, which comprises the sequence AGCGCAGAGGTAATAA (SEQ ID NO;

352);

u3) the nucleotide sequence of SEQ ID NO;

80, which comprises the sequence AGCGAATAGGTAAGTC (SEQ ID NO;

353);

v3) the nucleotide sequence of SEQ ID NO;

81, which comprises the sequence AGCGAATAGGTAAGTC (SEQ ID NO;

353);

w3) the nucleotide sequence of SEQ ID NO;

82, which comprises the sequence CGAGGGCAGGTAAGTA (SEQ ID NO;

354);

x3) the nucleotide sequence of SEQ ID NO;

83, which comprises the sequence CGAGGGCAGGTAAGTA (SEQ ID NO;

354);

y3) the nucleotide sequence of SEQ ID NO;

84, which comprises the sequence GGTGCCGAGGTAAGTT (SEQ ID NO;

355);

z3) the nucleotide sequence of SEQ ID NO;

85, which comprises the sequence GGTGCCGAGGTAAGTT (SEQ ID NO;

355);

a4) the nucleotide sequence of SEQ ID NO;

86, which comprises the sequence GGTGACTAGGTAAGTC (SEQ ID NO;

356);

b4) the nucleotide sequence of SEQ ID NO;

87, which comprises the sequence GGTGACTAGGTAAGTC (SEQ ID NO;

356);

c4) the nucleotide sequence of SEQ ID NO;

88, which comprises the sequence CGAGGGCAGGTAAGTT (SEQ ID NO;

357);

d4) the nucleotide sequence of SEQ ID NO;

89, which comprises the sequence CGAGGGCAGGTAAGTT (SEQ ID NO;

357);

e4) the nucleotide sequence of SEQ ID NO;

90, which comprises the sequence AGCGCAGAGGTAAGTA (SEQ ID NO;

358);

f4) the nucleotide sequence of SEQ ID NO;

91, which comprises the sequence AGCGCAGAGGTAAGTA (SEQ ID NO;

358);

g4) the nucleotide sequence of SEQ ID NO;

10 (657G);

h4) the nucleotide sequence of SEQ ID NO;

11 (657G);

i4) the nucleotide sequence of SEQ ID NO;

12 (658T);

j4) the nucleotide sequence of SEQ ID NO;

13 (658T);

k4) the nucleotide sequence of SEQ ID NO;

18 (S1+657G);

14) the nucleotide sequence of SEQ ID NO;

20 (S1+657G);

m4) the nucleotide sequence of SEQ ID NO;

19 (S1+658T);

n4) the nucleotide sequence of SEQ ID NO;

21 (S1+658T); and

o4) any combination of a1 through n4 above.

6. The cell of claim 1, wherein said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct comprises two or more second intronic nucleotide sequences.

7. The cell of claim 1, wherein said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct comprises two or more second intronic nucleotide sequences selected from the group consisting of:

a) second intronic nucleotide sequences in tandem within said first nucleotide sequence,

b) second intronic nucleotide sequences spaced at least 25 base pairs apart within said first nucleotide sequence,

c) second intronic nucleotide sequences spaced at least 50 base pairs apart within said first nucleotide sequence,

d) second intronic nucleotide sequences spaced at least 75 base pairs apart within said first nucleotide sequence,

e) second intronic nucleotide sequences spaced at least 100 base pairs apart within said first nucleotide sequence,

f) second intronic nucleotide sequences spaced at least 200 base pairs apart within said first nucleotide sequence,

g) second intronic nucleotide sequences spaced at least 300 base pairs apart within said first nucleotide sequence,

h) second intronic nucleotide sequences wherein a primary second intronic nucleotide sequence is located between a promoter and said first nucleotide sequence and a secondary second intronic nucleotide sequence is located within an open reading frame of said first nucleotide sequence; and

i) second intronic nucleotide sequences wherein a primary second intronic nucleotide sequence is located between said open reading frame and a poly A signal in said first nucleotide sequence and a secondary second intronic nucleotide sequence is located within said open reading frame of said first nucleotide sequence.

8. The cell of claim 6, wherein said two or more second intronic nucleotide sequences are the same within said first or second nucleic acid construct.

9. The cell of claim 6, wherein said two or more second intronic nucleotide sequences are each different from one another within said first or second nucleic acid construct.

10. The cell of claim 1, wherein said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct comprises two or more first nucleotide sequences.

11. The cell of claim 10, wherein said two or more first nucleotide sequences are the same within said first or second nucleic acid construct.

12. The cell of claim 10, wherein said two or more first nucleotide sequences are each different from one another with said first or second nucleic acid construct.

13. The cell of claim 1, wherein said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct further comprises a promoter that directs expression of said first nucleotide sequence(s) of said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct.

14. The cell of claim 13, wherein said second intronic nucleotide sequence of said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct is positioned between said promoter and said first nucleotide sequence(s).

15. The cell of claim 1, wherein said second intronic nucleotide sequence of said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct is positioned within an open reading frame of said first nucleotide sequence(s).

16. The cell of claim 1, wherein said second intronic nucleotide sequence of said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct is positioned within the 5′

one-third of an open reading frame of said first nucleotide sequence(s).

17. The cell of claim 1, wherein said second intronic nucleotide sequence of said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct is positioned within the middle one-third of said open reading frame of said first nucleotide sequence(s).

18. The cell of claim 1, wherein said second intronic nucleotide sequence of said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct is positioned within the 3′

one-third of said open reading frame of said first nucleotide sequence(s).

19. The cell of claim 1, wherein said second intronic nucleotide sequence of said first heterologous nucleic acid construct and/or said second heterologous nucleic acid construct is positioned between said open reading frame and a poly A signal in said first nucleotide sequence(s).

38. A composition comprising the cell of claim 1 in a pharmaceutically acceptable carrier.

40. A method of producing a functional RNA encoded by said first nucleotide sequence of said first heterologous nucleic acid construct or a functional RNA encoded by said.first nucleotide sequence of said second heterologous nucleic acid construct in the cell of claim 1, comprising:

introducing into said cell a blocking oligonucleotide and/or small molecule that blocks a member of said second set of splice elements of said second intronic nucleotide sequence of said first heterologous nucleic acid construct or of said second heterologous nucleic acid construct, thereby producing the functional RNA encoded by said first nucleotide sequence of said first heterologous nucleic acid construct or encoded by first nucleotide sequence of said second heterologous nucleic acid construct in said cell.

41. A method of producing a first functional RNA encoded by said first nucleotide sequence of said first heterologous nucleic acid construct and producing a second functional RNA encoded by said first nucleotide sequence of said second heterologous nucleic acid construct in the cell of claim 1, wherein said first functional RNA and said second functional RNA are different from each other, comprising:

a) introducing into said cell a first blocking oligonucleotide and/or first small molecule that blocks a member of said second set of splice elements of said second intronic nucleotide sequence of said first heterologous nucleic acid construct, thereby producing said first functional RNA encoded by said first nucleotide sequence of said first heterologous nucleic acid construct in said cell, and

b) introducing into said cell a second blocking oligonucleotide and/or second small molecule that blocks a member of said second set of splice elements of said second intronic nucleotide sequence of said second heterologous nucleic acid construct, thereby producing said second functional RNA encoded by said first nucleotide sequence of said second heterologous nucleic acid construct in said cell,

wherein said second intronic nucleotide sequence of said first heterologous nucleic acid construct and said second intronic nucleotide sequence of said second heterologous nucleic acid construct are different from each other and wherein said first blocking oligonucleotide and/or first small molecule and said second blocking oligonucleotide and/or second small molecule are different from each other.

44. The method of claim 40, wherein the blocking oligonucleotide does not activate RNase H.

45. The method of claim 40, wherein the blocking oligonucleotide comprises a modified internucleotide bridging phosphate residue selected from the group consisting of methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates, phosphoramidates and any combination thereof.

46. The method of claim 40, wherein the blocking oligonucleotide comprises a nucleotide having a loweralkyl substituent at the 2′

position thereof.

47. The method of claim 40, wherein the blocking oligonucleotide is from about eight to about 50 nucleotides in length.

48. The method of claim 40, wherein the cell is in a subject.

49. The method of claim 48, wherein the subject is a human.

20. An isolated nucleic acid comprising:

A) a first nucleotide sequence encoding a nucleotide sequence of interest (NOI); and

B) a second intronic nucleotide sequence operably associated with said first nucleotide sequence and wherein said second intronic nucleotide sequence is heterologous to said first nucleotide sequence and further wherein said second intronic nucleotide sequence is selected from the group consisting of;

a1) the nucleotide sequence of SEQ ID NO;

92 (S0 257 by intron);

b1) the nucleotide sequence of SEQ ID NO;

2 (S0−

GT);

c1) the nucleotide sequence of SEQ ID NO;

1 (S0−

CT);

d1) the nucleotide sequence of SEQ ID NO;

4 (S0−

GT+CT);

e1) the nucleotide sequence of SEQ ID NO;

3 (S1);

f1) the nucleotide sequence of SEQ ID NO;

5 (S1+CT);

g1) the nucleotide sequence of SEQ ID NO;

6 (M3);

h1) the nucleotide sequence of SEQ ID NO;

7 (M3+CT);

i1) the nucleotide sequence of SEQ ID NO;

8 (M6);

j1) the nucleotide sequence of SEQ ID NO;

9 (M6+CT);

k1) the nucleotide sequence of SEQ ID NO;

14 (M3+S1);

l1) the nucleotide sequence of SEQ ID NO;

16 (M3+S1+CT);

m1) the nucleotide sequence of SEQ ID NO;

15 (M6+S1);

n1) the nucleotide sequence of SEQ ID NO;

17 (M6+S1+CT);

o1) the nucleotide sequence of SEQ ID NO;

22, which comprises the sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉GTX₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO;

324), wherein X of any of X₁-X₉ and X₁₀-X₁₄ can be A, C, T or G, in any combination;

p1) the nucleotide sequence of SEQ ID NO;

23, which comprises the sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉GTX₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO;

324), wherein X of any of X₁-X₉ and X₁₀-X₁₄ can be A, C, T or G, in any combination;

q1) the nucleotide sequence of SEQ ID NO;

24, which comprises the sequence X₁GX₃GX₅X₆X₇AGGTAATAX₁₄ (SEQ ID NO;

325), wherein X of any of X₁, X₃-X₇ and X₁₄ can be A, C, T or G, in any combination;

r1) the nucleotide sequence of SEQ ID NO;

25, which comprises the sequence X₁GX₃GX₆X₆X₇AGGTAATAX₁₄ (SEQ ID NO;

325), wherein X of any of X₁, X₃-X₇ and X₁₄ can be A, C, T or G, in any combination;

s1) the nucleotide sequence of SEQ ID NO;

26, which comprises the sequence X₁GX₃GX₆X₆X₇AGGTAAGTX₁₄ (SEQ ID NO;

326), wherein X of any of X₁, X₃, X₅-X₇ and X₁₄ can be A, C, T or G, in any combination;

t1) the nucleotide sequence of SEQ ID NO;

27, which comprises the sequence X₁GX₃GX₆X₆X₇AGGTAAGTX₁₄ (SEQ ID NO;

326), wherein X of any of X₁, X₃, X₅-X₇ and X₁₄ can be A, C, T or G, in any combination;

u1) the nucleotide sequence of SEQ ID NO;

28, which comprises the sequence X₁X₂X₃X₄X₆X₆AAGGTAATAG (SEQ ID NO;

327), wherein X of any of X₁,-X₆ can be A, C, T or G, in any combination;

v1) the nucleotide sequence of SEQ ID NO;

29, which comprises the sequence X₁X₂X₃X₄X₆X₆AAGGTAATAG (SEQ ID NO;

327), wherein X of any of X₁,-X₆ can be A, C, T or G, in any combination;

w1) the nucleotide sequence of SEQ ID NO;

30, which comprises the sequence X₁GX₃X₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

328), wherein X of any of X₁ and X₃-X₇ can be A, C, T or G, in any combination;

x1) the nucleotide sequence of SEQ ID NO;

31, which comprises the sequence X₁GX₃X₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

328), wherein X of any of X₁, and X₃-X₇ can be A, C, T or G, in any combination;

y1) the nucleotide sequence of SEQ ID NO;

32, which comprises the sequence X₁X₂GX₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

329), wherein X of any of X₁, X₂ and X₄-X₇ can be A, C, T or G, in any combination;

z1) the nucleotide sequence of SEQ ID NO;

33, which comprises the sequence X₁X₂GX₄X₆X₆X₇AGGTAATAG (SEQ ID NO;

329), wherein X of any of X₁, X₂ and X₄-X₇ can be A, C, T or G, in any combination;

a2) the nucleotide sequence of SEQ ID NO;

34, which comprises the sequence X₁X₂X₃GX₆X₆X₇AGGTAATAG (SEQ ID NO;

330), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

b2) the nucleotide sequence of SEQ ID NO;

35, which comprises the sequence X₁X₂X₃GX₆X₆X₇AGGTAATAG (SEQ ID NO;

330), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

c2) the nucleotide sequence of SEQ ID NO;

36, which comprises the sequence X₁X₂X₃X₄TX₆X₇AGGTAATAG (SEQ ID NO;

331), wherein X of any of X₁-X₄ and X₆-X₇ can be A, C, T or G, in any combination;

d2) the nucleotide sequence of SEQ ID NO;

37, which comprises the sequence X₁X₂X₃X₄TX₈X₇AGGTAATAG (SEQ ID NO;

331), wherein X of any of X₁-X₄ and X₆-X₇ can be A, C, T or G, in any combination;

e2) the nucleotide sequence of SEQ ID NO;

38, which comprises the sequence X₁GGX₄X₅X₈X₇X₈GGTAATAG (SEQ ID NO;

332), wherein X of any of X₁ and X₄-X₈ can be A, C, T or G, in any combination;

f2) the nucleotide sequence of SEQ ID NO;

39, which comprises the sequence X₁GGX₄X₅X₈X₇X₈GGTAATAG (SEQ ID NO;

332), wherein X of any of X₁ and X₄-X₅ can be A, C, T or G, in any combination;

g2) the nucleotide sequence of SEQ ID NO;

40, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAATAG (SEQ ID NO;

333), wherein X of any of X₁, X₂ and X₅-X₈ can be A, C, T or G, in any combination;

h2) the nucleotide sequence of SEQ ID NO;

41, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAATAG (SEQ ID NO;

333), wherein X of any of X₁, X₂ and X₅-X₈ can be A, C, T or G, in any combination;

i2) the nucleotide sequence of SEQ ID NO;

42, which comprises the sequence X₁X₂X₃GTX₈X₇X₈GGTAATAG (SEQ ID NO;

334), wherein X of any of X₁-X₃ and X₆-X₈ can be A, C, T or G, in any combination;

j2) the nucleotide sequence of SEQ ID NO;

43, which comprises the sequence X₁X₂X₃GTX₈X₇X₈GGTAATAG (SEQ ID NO;

334), wherein X of any of X₁-X₃ and X₆-X₈ can be A, C, T or G, in any combination;

k2) the nucleotide sequence of SEQ ID NO;

44, which comprises the sequence X₁X₂X₃X₄TTX₇X₈GGTAATAG (SEQ ID NO;

335), wherein X of any of X₁-X₄ and X₇-X₈ can be A, C, T or G, in any combination;

l2) the nucleotide sequence of SEQ ID NO;

45, which comprises the sequence X₁X₂X₃X₄TTX₇X₈GGTAATAG (SEQ ID NO;

335), wherein X of any of X₁-X₄ and X₇-X₈ can be A, C, T or G, in any combination;

m2) the nucleotide sequence of SEQ ID NO;

46, which comprises the sequence X₁X₂X₃X₄X₅TAX₈GGTAATAG (SEQ ID NO;

336), wherein X of any of X₁-X₅ and X₈ can be A, C, T or G, in any combination;

n2) the nucleotide sequence of SEQ ID NO;

47, which comprises the sequence X₁X₂X₃X₄X₅TAX₈GGTAATAG (SEQ ID NO;

336), wherein X of any of X₁-X₅ and X_s can be A, C, T or G, in any combination;

o2) the nucleotide sequence of SEQ ID NO;

48, which comprises the sequence X₁X₂X₃X₄X₅X₈AAGGTAAGTG (SEQ ID NO;

337), wherein X of any of X₁-X₆ can be A, C, T or G, in any combination;

p2) the nucleotide sequence of SEQ ID NO;

49, which comprises the sequence X₁X₂X₃X₄X₅X₆AAGGTAAGTG (SEQ ID NO;

337), wherein X of any of X₁-X₆ can be A, C, T or G, in any combination;

q2) the nucleotide sequence of SEQ ID NO;

50, which comprises the sequence X₁GX₃X₄X₅X₈X₇AGGTAAGTG (SEQ ID NO;

338), wherein X of any of X₁ and X₃-X₇ can be A, C, T or G, in any combination;

r2) the nucleotide sequence of SEQ ID NO;

51, which comprises the sequence X₁GX₃X₄X₅X₆X₇AGGTAAGTG (SEQ ID NO;

338), wherein X of any of X₁ and X₃-X₇ can be A, C, T or G, in any combination;

s2) the nucleotide sequence of SEQ ID NO;

52, in any combination, which comprises the sequence X₁X₂GX₄X₅X₈X₇AGGTAAGTG (SEQ ID NO;

339), wherein X of any of X₁-X₂ and X₄-X₇ can be A, C, T or G;

t2) the nucleotide sequence of SEQ ID NO;

53, which comprises the sequence X₁X₂GX₄X₅X₆X₇AGGTAAGTG (SEQ ID NO;

339), wherein X of any of X₁-X₂ and X₄-X₇ can be A, C, T or G, in any combination;

u2) the nucleotide sequence of SEQ ID NO;

54, which comprises the sequence X₁X₂X₃GX₅X₈X₇AGGTAAGTG (SEQ ID NO;

340), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

v2) the nucleotide sequence of SEQ ID NO;

55, which comprises the sequence X₁X₂X₃GX₅X₈X₇AGGTAAGTG (SEQ ID NO;

340), wherein X of any of X₁-X₃ and X₅-X₇ can be A, C, T or G, in any combination;

w2) the nucleotide sequence of SEQ ID NO;

56, which comprises the sequence X₁X₂X₃X₄TX₆X₇AGGTAAGTG (SEQ ID NO;

341), wherein X of any of X₁-X₄ and X₆-X₇ can be A, C, T or G, in any combination;

x2) the nucleotide sequence of SEQ ID NO;

57, which comprises the sequence X₁X₂X₃X₄TX₈X₇AGGTAAGTG (SEQ ID NO;

341), wherein X of any of X₁-X₄ and X₆-X₇ can be A, C, T or G, in any combination;

y2) the nucleotide sequence of SEQ ID NO;

58, which comprises the sequence X₁GGX₄X₅X₈X₇X₈GGTAAGTG (SEQ ID NO;

342), wherein X of any of X₁ and X₄-X₈ can be A, C, T or G, in any combination;

z2) the nucleotide sequence of SEQ ID NO;

59, which comprises the sequence X₁GGX₄X₅X₆X₇X₈GGTAAGTG (SEQ ID NO;

342), wherein X of any of X₁ and X₄-X₈ can be A, C, T or G, in any combination;

a3) the nucleotide sequence of SEQ ID NO;

60, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAAGTG (SEQ ID NO;

343), wherein X of any of X₁-X₂ and X₅-X₈ can be A, C, T or G, in any combination;

b3) the nucleotide sequence of SEQ ID NO;

61, which comprises the sequence X₁X₂GGX₅X₈X₇X₈GGTAAGTG (SEQ ID NO;

343), wherein X of any of X₁-X₂ and X₅-X₈ can be A, C, T or G, in any combination;

c3) the nucleotide sequence of SEQ ID NO;

62, which comprises the sequence X₁X₂X₃GTX₈X₇X₈GGTAAGTG (SEQ ID NO;

344), wherein X of any of X₁-X₂ and X₅-X₈ can be A, C, T or G, in any combination;

d3) the nucleotide sequence of SEQ ID NO;

63, which comprises the sequence X₁X₂X₃GTX₈X₇X₈GGTAAGTG (SEQ ID NO;

344), wherein X of any of X₁-X₃ and X₆-X₈ can be A, C, T or G, in any combination;

e3) the nucleotide sequence of SEQ ID NO;

64, which comprises the sequence X₁X₂X₃X₄TTX₇X₈GGTAAGTG (SEQ ID NO;

345), wherein X of any of X₁-X₄ and X₇-X₈ can be A, C, T or G, in any combination;

f3) the nucleotide sequence of SEQ ID NO;

65, which comprises the sequence X₁X₂X₃X_ITTX₇X₈GGTAAGTG (SEQ ID NO;

345), wherein X of any of X₁-X₁and X₇-X₈ can be A, C, T or G, in any combination;

g3) the nucleotide sequence of SEQ ID NO;

66, which comprises the sequence X₁X₂X₃X₄X₅TAX₈GGTAATGTG (SEQ ID NO;

346), wherein X of any of X₁-X₅ and X₅ can be A, C, T or G, in any combination;

h3) the nucleotide sequence of SEQ ID NO;

67, which comprises the sequence X₁X₂X₃X₄X₅TAX₈GGTAATGTG (SEQ ID NO;

346), wherein X of any of X₁-X₅ and X₈ can be A, C, T or G, in any combination;

i3) the nucleotide sequence of SEQ ID NO;

68, which comprises the sequence AGCGAATAGGTAATAC (SEQ ID NO;

347);

j3) the nucleotide sequence of SEQ ID NO;

69, which comprises the sequence AGCGAATAGGTAATAC (SEQ ID NO;

347);

k3) the nucleotide sequence of SEQ ID NO;

70, which comprises the sequence CGAGGGCAGGTAATAA (SEQ ID NO;

348);

l3) the nucleotide sequence of SEQ ID NO;

71, which comprises the sequence CGAGGGCAGGTAATAA (SEQ ID NO;

348);

m3) the nucleotide sequence of SEQ ID NO;

72, which comprises the sequence GGTGCCGAGGTAATAT (SEQ ID NO;

349);

n3) the nucleotide sequence of SEQ ID NO;

73, which comprises the sequence GGTGCCGAGGTAATAT (SEQ ID NO;

349);

o3) the nucleotide sequence of SEQ ID NO;

74, which comprises the sequence GGTGACTAGGTAATAC (SEQ ID NO;

350);

p3) the nucleotide sequence of SEQ ID NO;

75, which comprises the sequence GGTGACTAGGTAATAC (SEQ ID NO;

350);

q3) the nucleotide sequence of SEQ ID NO;

76, which comprises the sequence CGAGGGCAGGTAATAT (SEQ ID NO;

351);

r3) the nucleotide sequence of SEQ ID NO;

77, which comprises the sequence CGAGGGCAGGTAATAT (SEQ ID NO;

351);

s3) the nucleotide sequence of SEQ ID NO;

78, which comprises the sequence AGCGCAGAGGTAATAA (SEQ ID NO;

352);

t3) the nucleotide sequence of SEQ ID NO;

79, which comprises the sequence AGCGCAGAGGTAATAA (SEQ ID NO;

352);

u3) the nucleotide sequence of SEQ ID NO;

80, which comprises the sequence AGCGAATAGGTAAGTC (SEQ ID NO;

353);

v3) the nucleotide sequence of SEQ ID NO;

81, which comprises the sequence AGCGAATAGGTAAGTC (SEQ ID NO;

353);

w3) the nucleotide sequence of SEQ ID NO;

82, which comprises the sequence CGAGGGCAGGTAAGTA (SEQ ID NO;

354);

x3) the nucleotide sequence of SEQ ID NO;

83, which comprises the sequence CGAGGGCAGGTAAGTA (SEQ ID NO;

354);

y3) the nucleotide sequence of SEQ ID NO;

84, which comprises the sequence GGTGCCGAGGTAAGTT (SEQ ID NO;

355);

z3) the nucleotide sequence of SEQ ID NO;

85, which comprises the sequence GGTGCCGAGGTAAGTT (SEQ ID NO;

355);

a4) the nucleotide sequence of SEQ ID NO;

86, which comprises the sequence GGTGACTAGGTAAGTC (SEQ ID NO;

356);

b4) the nucleotide sequence of SEQ ID NO;

87. which comprises the sequence GGTGACTAGGTAAGTC (SEQ ID NO;

356);

c4) the nucleotide sequence of SEQ ID NO;

88, which comprises the sequence CGAGGGCAGGTAAGTT (SEQ ID NO;

357);

d4) the nucleotide sequence of SEQ ID NO;

89, which comprises the sequence CGAGGGCAGGTAAGTT (SEQ ID NO;

357);

e4) the nucleotide sequence of SEQ ID NO;

90, which comprises the sequence AGCGCAGAGGTAAGTA (SEQ ID NO;

358);

f4) the nucleotide sequence of SEQ ID NO;

91, which comprises the sequence AGCGCAGAGGTAAGTA (SEQ ID NO;

358);

g4) the nucleotide sequence of SEQ ID NO;

10 (657G);

h4) the nucleotide sequence of SEQ ID NO;

11 (657G);

i4) the nucleotide sequence of SEQ ID NO;

12 (658T);

j4) the nucleotide sequence of SEQ ID NO;

13 (658T);

k4) the nucleotide sequence of SEQ ID NO;

18 (51+657G);

l4) the nucleotide sequence of SEQ ID NO;

20 (S1+657G);

m4) the nucleotide sequence of SEQ ID NO;

19 (S1+658T);

n4) the nucleotide sequence of SEQ ID NO;

21 (S1+658T); and

o4) any combination of a1 through n4 above.

21. The isolated nucleic acid of claim 20, comprising two or more second intronic nucleotide sequences.

22. The isolated nucleic acid of claim 20, comprising two or more second intronic nucleotide sequences selected from the group consisting of:

a) second intronic nucleotide sequences in tandem within said first nucleotide sequence,

b) second intronic nucleotide sequences spaced at least 25 base pairs apart within said first nucleotide sequence,

c) second intronic nucleotide sequences spaced at least 50 base pairs apart within said first nucleotide sequence,

d) second intronic nucleotide sequences spaced at least 75 base pairs apart within said first nucleotide sequence,

e) second intronic nucleotide sequences spaced at least 100 base pairs apart within said first nucleotide sequence,

f) second intronic nucleotide sequences spaced at least 200 base pairs apart within said first nucleotide sequence,

g) second intronic nucleotide sequences spaced at least 300 base pairs apart within said first nucleotide sequence,

h) second intronic nucleotide sequences wherein a primary second intronic nucleotide sequence is located between a promoter and an open reading frame of said first nucleotide sequence and a secondary second intronic nucleotide sequence is located within an open reading frame of said first nucleotide sequence; and

i) second intronic nucleotide sequences wherein a primary second intronic nucleotide sequence is located between an open reading frame and a poly A signal in said first nucleotide sequence and a secondary second intronic nucleotide sequence is located within said open reading frame of said first nucleotide sequence.

23. The nucleic acid of claim 20, wherein said first nucleotide sequence encodes a nucleotide sequence of interest (NOI) selected from the group consisting of:

a) a nucleotide sequence encoding a protein or peptide;

b) a nucleotide sequence encoding a product having activity as an interfering RNA;

c) a nucleotide sequence encoding a product having enzymatic activity as an RNA;

d) a nucleotide sequence encoding a ribozyme;

e) a nucleotide sequence encoding an antisense sequence;

f) a nucleotide sequence encoding a small nuclear RNA (snRNA); and

g) any combination of (a)-(f) above

24. The nucleic acid of claim 20, comprising two or more first nucleotide sequences.

25. The nucleic acid of claim 24, wherein said two or more first nucleotide sequences are the same.

26. The nucleic acid of claim 24, wherein said two or more first nucleotide sequences are each different from one another.

27. The nucleic acid of claim 20, further comprising a promoter that directs expression of said first nucleotide sequence.

28. The nucleic acid of claim 27, wherein said second intronic nucleotide sequence is positioned between the promoter and an open reading frame of said first nucleotide sequence.

29. The nucleic acid of claim 20, wherein said second intronic nucleotide sequence is positioned within said open reading frame of said first nucleotide sequence.

30. The nucleic acid of claim 29, wherein said second intronic nucleotide sequence is positioned within the 5′

one-third of said open reading frame of said first nucleotide sequence.

31. The nucleic acid of claim 29, wherein said second intronic nucleotide sequence is positioned within the middle one-third of said open reading frame of said first nucleotide sequence.

32. The nucleic acid of claim 29, wherein said second intronic nucleotide sequence is positioned within the 3′

one-third of said open reading frame of said first nucleotide sequence.

33. The nucleic acid of claim 20, wherein said second intronic nucleotide sequence is positioned between said open reading frame and a poly A signal in said first nucleotide sequence.

34. A vector comprising the nucleic acid of claim 20.

35. A cell comprising the nucleic acid of claim 20.

36. A cell comprising the vector of claim 34.

37. A composition comprising the nucleic acid of claim 20 in a pharmaceutically acceptable carrier.

39. A composition comprising the vector of claim 34 in a pharmaceutically acceptable carrier.

42. A method for producing a functional RNA encoded by said first nucleotide sequence of said isolated nucleic acid of claim 20, comprising contacting a blocking oligonucleotide and/or small molecule with the isolated nucleic acid under conditions that permit splicing, wherein the blocking oligonucleotide and/or small molecule blocks a member of said second set of splice elements of said second intronic nucleotide sequence, thereby producing said functional RNA encoded by said first nucleotide sequence.

43. The method of claim 42, wherein the blocking oligonucleotide and/or small molecule is introduced into a cell comprising the isolated nucleic acid.