Enhanced biotherapeutic production using inhibitory RNA
Inventors
LaDine, James R. • Marshall, William S. • Fedorov, Yuriy • Yamada, Christina
Assignees
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Publication Number
US-8273722-B2
Publication Date
2012-09-25
Expiration Date
Abstract
Compositions, kits, systems, equipment, and protocols utilize synthetic siRNA having a delivery facilitating moiety in improved bioprocesses that enhance the production of biomaterials. The siRNA can target genes associated with the following: 1) deleterious vector derived genes; 2) genes that confer non-optimal growth or differentiation properties to the cells; 3) genes that can influence heterogeneity or post-translational modification pattern of the desirable gene product; 4) genes that highly express non-desired proteins; 5) genes that express proteins which interfere with purification of the desired protein; and 6) other genes that can interfere with the bioprocess.
Core Innovation
The invention relates to producing a protein-containing product from CHO cells by culturing recombinant host CHO cells containing nucleic acid encoding at least a portion of the protein-containing product, and contacting the recombinant host CHO cell with a synthetic, chemically modified siRNA. The contacting is performed under conditions in which the synthetic siRNA passively transfects the recombinant host CHO cell in an amount sufficient to inhibit expression of a first protein, where expression of the first protein decreases the production efficiency of the protein-containing product.
The synthetic siRNA includes a sense strand with 2'-O-methyl modifications on the ultimate and penultimate nucleotides and on each pyrimidine nucleotide, and a cholesterol or cholesterol derivative conjugate coupled to the 3' end of the sense strand through a linker having a 3-member to 8-member carbon chain. The antisense strand includes 2'-fluorine modifications on each pyrimidine nucleotide, a 5' terminal phosphate, and a U-U 3' overhang that includes phosphorothioate internucleotide linkages.
The antisense strand further includes one of an A-A mismatch, a U-U mismatch, a C-C mismatch, or a G-G mismatch between nucleotide 6 from the 5' end of the sense strand and the opposite nucleotide on the antisense strand. Expression of the first protein is inhibited, followed by incubating under conditions that express the protein-containing product and recovering the protein-containing product.
Claims Coverage
The provided excerpt identifies two independent method claims. Both independent claims share the core requirement of passively transfecting with a synthetic, chemically modified siRNA linked to a delivery facilitating moiety to inhibit a first protein whose expression decreases production efficiency, with the host-cell context differing as CHO cells versus recombinant host cell.
Passively transfecting recombinant CHO cells with synthetic chemically modified siRNA linked to a delivery conjugate to inhibit a first protein
Culturing recombinant host CHO cells containing nucleic acid encoding at least a portion of the protein-containing product, contacting the recombinant host CHO cell with a synthetic, chemically modified siRNA having a delivery facilitating conjugate linked to a polynucleotide of the siRNA under conditions in which the synthetic siRNA passively transfects the recombinant host CHO cell in an amount sufficient to inhibit expression of a first protein, where expression of the first protein decreases the production efficiency of the protein-containing product, followed by incubating to express the protein-containing product and recovering the protein-containing product.
Passively transfecting a recombinant host cell with synthetic chemically modified siRNA having a cholesterol-linked sense strand and specified antisense chemistry
Culturing the recombinant host cell containing nucleic acid encoding at least a portion of the protein-containing product, contacting the recombinant host cell with a synthetic, chemically modified siRNA having a delivery facilitating moiety linked to a polynucleotide of the siRNA under conditions in which the synthetic siRNA passively transfects the recombinant host cell in an amount sufficient to inhibit expression of a first protein, where expression of the first protein decreases the production efficiency of the protein-containing product, followed by incubating to express the protein-containing product and recovering the protein-containing product.
Both independent claims cover methods that rely on passive transfection of recombinant cells with synthetic, chemically modified siRNA containing a cholesterol or cholesterol-derivative conjugate and specific sense/antisense chemical modifications and a defined mismatch position, to inhibit expression of a first protein that decreases production efficiency, enabling subsequent expression and recovery of a protein-containing product.
Stated Advantages
Inhibiting expression of the first protein is associated with producing a protein-containing product with improved production efficiency, as expression of the first protein decreases production efficiency.
Documented Applications
Production of a protein-containing product from recombinant host CHO cells by culturing cells encoding the protein, passively transfecting with chemically modified siRNA to inhibit expression of a first protein, then expressing and recovering the protein-containing product.
Examples in CHO cells where modified/conjugated siRNA achieves target protein knockdown, modulates apoptosis phenotypes, and increases secreted tPA production.
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