Enhanced biotherapeutic production using inhibitory RNA

1. A method for producing a protein-containing product from CHO cells, the method comprising:

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, said contacting being 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,

wherein the synthetic, chemically modified siRNA includes:

a sense strand having:

2′-O-methyl modifications on the ultimate and penultimate nucleotides;

2′-O-methyl modifications 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; and

an antisense strand having:

2′-fluorine modifications on each pyrimidine nucleotide a 5′ terminal phosphate;

a U-U 3′ overhang that includes phosphorothioate internucleotide linkages; and

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,

wherein expression of the first protein decreases the production efficiency of the protein-containing product;

incubating the recombinant host CHO cells with the synthetic siRNA under conditions that inhibit expression of the first protein;

incubating the recombinant host CHO cells under conditions that express the protein-containing product; and

recovering the protein-containing product.

2. The method of claim 1 , wherein the delivery facilitating conjugate is a lipid.

3. The method of claim 2 , wherein the lipid is cholesterol or derivative thereof.

4. The method of claim 1 , further comprising:

inhibiting expression of the first protein during a first portion of the step of incubating the recombinant host cell while the recombinant host cell is replicating; and

expressing the protein-containing product during a second portion of incubating the recombinant host cell after the expression of the first protein has been inhibited.

5. The method of claim 1 , wherein the first protein is the protein of the protein-containing product expressed at a time point unfavorable for producing a protein-containing product.

6. The method of claim 1 , wherein the first protein is selected from at least one of the following:

a vector protein that is deleterious to cell viability;

a vector protein that is deleterious to production of the protein-containing product;

a protein that is expressed at a particular stage of a bioprocess that is deleterious to the growth of the recombinant host cell;

a protein that is expressed in Phase I of a bioprocess;

a protein that is expressed in Phase II of a bioprocess;

a protein that is expressed in Phase III of a bioprocess;

a protein that modifies the protein-containing product into an undesired form;

a protease; a glycosylase; a kinase;

a phosphatase; a protein that post-translationally modifies the protein-containing product;

a protein that is over-expressed; a protein that is highly expressed so as to be deleterious to the expression of the protein-containing product;

a protein that causes degradation of the protein-containing product;

a ubiqutinase;

a protein that associates with the protein-containing product;

a protein that has substantially the molecular weight of the protein-containing product;

a protein that has substantially the isoelectric point of the protein-containing product;

a protein that has substantially the purification properties of the protein-containing product;

a protein that interferes with recovering the protein-containing product;

a protein that co-elutes with the protein-containing product;

a viral protein;

a protein having an activity that introduces a substantial structural heterogeneity into the protein-containing product;

a fucosyl transferase;

a caspase;

a calcium transporter; or

CD16(Fc)(RIII).

7. The method of claim 1 , wherein the synthetic siRNA contacts the recombinant host CHO cell prior to incubating the recombinant host cell in a bioreactor.

8. The method of claim 1 , wherein the recombinant host CHO cell is incubated for a first time period in a bioreactor, is then contacted with the synthetic siRNA, and is then incubated for a second time period in a bioreactor.

9. The method of claim 1 , further comprising:

incubating the recombinant host CHO cell for a first time period in a bioreactor in the absence of the synthetic siRNA;

contacting with the synthetic siRNA in a bioreactor; and

incubating the recombinant host CHO cell for a second time period in a bioreactor.

10. The method of claim 1 , further comprising inhibiting the production of the first protein so as to enhance bioproduction of the protein-containing product compared to when production of the first protein is not inhibited.

11. The method of claim 1 , wherein the method comprises at least one of the following:

inhibiting production of fucosyl transferase;

inhibiting production of caspases to inhibit apoptosis;

silencing cell cycle progression genes to inhibit cell proliferation;

silencing pro-senescence genes to inhibit host cell death;

silencing calcium transporter genes to limit cross links;

silencing a gene encoding CD16(Fc)(RIII) to prohibit aggregation;

silencing genes encoding factors that destabilize transcriptions and/or affect translation; or

silencing genes encoding factors associated with glycosylation of proteins.

12. The method of claim 1 , wherein the protein-containing product is recovered from the recombinant host CHO cell.

13. The method of claim 1 , wherein the protein-containing product is exported from the recombinant host CHO cell to the surrounding media during the incubation of the recombinant host cell, and the protein-containing product is recovered from the surrounding media.

14. A method for producing a protein-containing product from a recombinant host cell, the method comprising:

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, said contacting being under conditions in which the synthetic siRNA passively transfects the recombinant host cell in an amount sufficient to inhibit expression of a first protein,

wherein the synthetic, chemically modified siRNA includes:

a sense strand having:

2′-O-methyl modifications on the ultimate and penultimate nucleotides;

2′-O-methyl modifications 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; and

an antisense strand having:

2′-fluorine modifications on each pyrimidine nucleotide a 5′ terminal phosphate;

a U-U 3′ overhang that includes phosphorothioate internucleotide linkages; and

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,

wherein expression of the first protein decreases the production efficiency of the protein-containing product; incubating the recombinant host cell with the synthetic siRNA under conditions that inhibit expression of the first protein;

incubating the recombinant host cell under conditions that express the protein-containing product; and

recovering the protein-containing product.

15. The method of claim 14 wherein the recombinant host cell is a CHO cell.

16. The method of claim 14 wherein the delivery facilitating moiety conjugate is a lipid.

17. The method of claim 16 wherein the lipid is cholesterol or derivative thereof.