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US 11,162,102 B2Granted
Modifications of mammalian cells using artificial micro-RNA to alter their properties and the compositions of their products
AssigneeDna Twopointo Inc.
InventorsJeremy Minshull, Maggie Lee, Varsha Sitaraman, Ferenc Boldog
Life of this patent
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Abstract
The present invention provides methods and compositions for stable genetic modification of cultured mammalian cells. The genetic modifications can be used to produce cultured mammalian cells for therapeutic or diagnostic purposes.
Claims24
A polynucleotide comprising
- a) a segment encoding a multi-hairpin amiRNA sequence, wherein the segment comprises
- i) a first guide strand sequence comprising a contiguous 19 nucleotide sequence that is perfectly complementary to a first target site in a natural mammalian cellular mRNA of SEQ ID NO:
- 21 and a first passenger strand sequence comprising a contiguous 19 nucleotide sequence that is at least 78% complementary to the first guide strand sequence, wherein the first guide strand and first passenger strand sequence are separated by between 5 and 35 nucleotides;
- ii) a second guide strand sequence comprising a contiguous 19 nucleotide sequence that is perfectly complementary to a second target site different than the first target site in the same natural mammalian cellular mRNA as the first guide strand sequence and a second passenger strand sequence comprising a contiguous 19 nucleotide sequence that is at least 78% complementary to the second guide strand sequence, wherein the second guide strand and second passenger strand sequences are separated by between 5 and 35 nucleotides, and wherein the first and second guide strand sequences are different from each other;
- and
- b) a eukaryotic promoter that is active in a mammalian cell and is transcribed by RNA polymerase II or RNA polymerase III operably linked to the segment encoding the amiRNA sequence, wherein the amiRNA sequence can be expressed and fold into multiple hairpins;
- wherein the first and second guide strand sequences are selected from SEQ ID NOs:
- 114-129.
5 more elements
A polynucleotide comprising
- a) a segment encoding a multi-hairpin amiRNA sequence, wherein the segment comprises
- i) a first guide strand sequence comprising a contiguous 19 nucleotide sequence that is perfectly complementary to a first target site in a natural mammalian cellular mRNA of SEQ ID NO:
- 23 and a first passenger strand sequence comprising a contiguous 19 nucleotide sequence that is at least 78% complementary to the first guide strand sequence, wherein the first guide strand and first passenger strand sequence are separated by between 5 and 35 nucleotides;
- ii) a second guide strand sequence comprising a contiguous 19 nucleotide sequence that is perfectly complementary to a second target site different than the first target site in the same natural mammalian cellular mRNA as the first guide strand sequence and a second passenger strand sequence comprising a contiguous 19 nucleotide sequence that is at least 78% complementary to the second guide strand sequence, wherein the second guide strand and second passenger strand sequences are separated by between 5 and 35 nucleotides, and wherein the first and second guide strand sequences are different from each other;
- and
- b) a eukaryotic promoter that is active in a mammalian cell and is transcribed by RNA polymerase II or RNA polymerase III operably linked to the segment encoding the amiRNA sequence, wherein the amiRNA sequence can be expressed and fold into multiple hairpins;
- wherein the first and second guide strand sequences are selected from SEQ ID NOS:130-142.
4 more elements
Dependencies are inferred from the claim text.