Multi-microRNA methods and compositions
View Patent ↗Provided are DNAs comprising a polynucleotide that encodes at least a first modified miR- 30 precursor and a second modified miR- 30 precursor. Also provided are vectors comprising the the DNAs, where the vector can replicate in a host cell. Additionally, specific lentiviral vectors comprising the above-described DNA are provided, as are methods of inhibiting expression of a target gene in a eukaryotic cell.
1. A DNA comprising a polynucleotide that encodes at east a first modified miR-30 precursor and a second modified miR-30 precursor,
wherein the first and second modified miR-30 precursors are each at least 80 nucleotides long and comprise a stem-loop structure,
wherein the first modified miR-30 precursor further comprises a first miRNA sequence of 20-22 nucleotides on the stem of the stem-loop structure complementary to a portion of a first target sequence wherein the first target sequence is a first naturally occurring mRNA sequence,
wherein the second modified miR-30 precursor further comprises a second miRNA sequence of 20-22 nucleotides on the stem-loop structure complementary to a second target sequence, and
wherein the first and second modified miR-30 precursors each independently further comprise a sequence complementary to the first and second miRNA sequence of 20-22 nucleotides, respectively, that base-pairs to the first and second miRNA sequence of 20-22 nucleotides, respectively, in the stem of the stem-loop structure, and wherein the first modified miR-30 precursor and the second modified miR-30 precursor are 13-25 nucleotides apart.
2. The DNA of claim 1 , wherein the first target sequence is the same as the second target sequence.
3. The DNA of claim 1 , wherein the first target sequence and he second target sequence are on the same mRNA or on different mRNAs.
4. The DNA of claim 1 , wherein the second target sequence does not natural y occur in the same species as the first target sequence.
5. The DNA of claim 1 , wherein the second target sequence naturally occurs in the same species as the first target sequence.
6. The DNA of claim 1 , wherein the modified miR-30 comprises SEQ ID NO:1.
7. The DNA of claim 1 , wherein the modified miR-30 comprises SEQ ID NO:2.
8. The DNA of claim 1 , wherein the modified miR-30 comprises SEQ ID NO:3.
9. The DNA of claim 1 , wherein the first target sequence and/or the second target sequence is complementary to a DNA from a mammalian pathogen.
10. The DNA of claim 9 , wherein the mammalian pathogen is a human pathogen.
11. The DNA of claim 9 , wherein the mammalian pathogen is a virus.
12. The DNA of claim 1 , wherein the first target sequence and/or the second target sequence is complementary to a mammalian DNA.
13. The DNA of claim 12 , wherein the mammalian mRNA is a human mRNA.
14. The DNA of claim 12 , wherein the mammalian mRNA encodes an enzyme.
15. The DNA of claim 14 , wherein the enzyme is a kinase or phosphatase.
16. The DNA of claim 12 , wherein the mammalian mRNA encodes a skp2 protein.
17. The DNA of claim 12 , wherein the mammalian mRNA encodes a receptor.
18. The DNA of claim 1 , wherein the first and second modified miR-30 precursors are each at least 85 nucleotides long.
19. The DNA of claim 1 , wherein the first and second modified miR-30 precursors are each 80-150 nucleotides long.
20. The DNA of claim 1 , further comprising a; site for a restriction endonuclease between the firs modified miR-30 precursor and the second modified miR-30 precursor.
21. The DNA of claim 1 , further comprising a promoter operably linked to the isolated DNA such that, when introduced into a eukaryotic cell, the first miR-30 precursor is transcribed and processed into the first miRNA and the second miR-30 precursor is transcribed and processed into the second miRNA.
22. The DNA of claim 21 , wherein the promoter is a mammalian promoter.
23. The DNA of claim 21 , wherein expression of a polypeptide encoded by the first naturally occurring mRNA sequence is reduced in the eukaryotic cell after the isolated DNA is introduced into the eukaryotic cell.
24. The DNA of claim 1 , further comprising a gene encoding a detectable moiety.
25. The DNA of claim 24 , wherein the gene encoding a detectable moiety encodes a green fluorescent protein (GFP).
26. The DNA of claim 1 , wherein
the first and second modified miR-30 precursors are each about 118 nucleotides long;
the first modified miR-30 precursor and the second modified miR-30 precursor are about 18 nucleotides apart;
the DNA further comprises a site for a restriction endonuclease between the first modified miR-30 precursor and the second modified miR-30 precursor;
the DNA further comprises a gene encoding a encodes a green fluorescent protein (GFP); and
the DNA further comprises a promoter operably linked to the isolated DNA such that, when introduced into a eukaryotic cell, the first miR-30 precursor is transcribed and processed into the first miRNA and the second miR-30 precursor is transcribed and processed into the second miRNA, wherein the promoter is a cytomegalovirus promoter.
27. The DNA of claim 26 , wherein the first target sequence and/or the second target sequence is complementary to a mammalian mRNA encoding a skp2 protein.
28. A eukaryotic cell comprising the DNA of claim 21 .
29. A vector comprising the DNA of claim 21 , wherein the vector can replicate in a host cell.
30. A lentiviral vector comprising the isolated DNA of claim 26 .
31. A mammalian cell transfected with the lentiviral vector of claim 30 .
32. A method of inhibiting expression of a target gene in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a DNA comprising a polynucleotide that encodes at least a first modified miR-30 precursor and a second modified miR-30 precursor,
wherein the first and second modified miR-30 precursors are each at least 80 nucleotides long and comprise a stem-loop structure,
wherein the first modified miR-30 precursor further comprises a first miRNA sequence of 20-22 nucleotides on the stem of the stem-loop structure complementary to a portion of a first target sequence, wherein the first target sequence is the mRNA of the target gene,
wherein the second modified miR-30 precursor further comprises a second miRNA sequence of 20-22 nucleotides on the stem-loop structure complementary to a second, target sequence,
wherein the first and second modified miR-30 precursors each independently further comprise a sequence complementary to the first and second miRNA sequence of 20-22 nucleotides, respectively, that base-pairs to the first and second miRNA sequence of 20-22 nucleotides, respectively, in the miR-30 precursor in the stem of the stem-loop structure,
wherein the first modified miR-30 precursor and the second modified miR-30 precursor are 13-25 nucleotides apart, and
wherein the DNA further comprises a promoter operably linked to the DNA such that, when the DNA is introduced into the eukaryotic cell, the first miR-30 precursor is transcribed and processed into the first miRNA and the second miR-30 precursor is transcribed and processed into the second miRNA, so as to thereby inhibit expression of the target gene in the eukaryotic cell.