title: Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt- and actin-resistant variant of human deoxyribonuclease I in CHO cells.
abstract: While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin. Engineered versions of hDNase I, bearing multiple point mutations, which renders them Hyperactive, Salt- and Actin-Resistant (HSAR-hDNase I) have been developed previously. However, constitutive expression of HSAR-hDNase I enzymes has been very challenging and, despite considerable efforts and screening thousands of clones, no stable clone capable of constitutive expression had been obtained. Here, we developed a regulated expression system for stable expression of an HSAR-hDNase I in Chinese Hamster Ovary (CHO) cells. The HSAR-hDNase I clones were stable and, upon induction, expressed enzymatically functional protein. Our findings suggest that degradation of host's DNA mediated by HSAR-hDNase I during cell division is the likely cause of clonal instability observed in cells constitutively expressing this protein. Purified HSAR-hDNase I was both hyperactive and resistant to inhibition by salt and G-actin, resulting in an enzyme having ca. 10-fold greater specific activity and the potential to be a superior therapeutic agent to wild type (WT) hDNase I. Furthermore, the ability to regulate hDNase I expression has enabled process development improvements that achieve higher cell growth and product titers while maintaining product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 32:523-533, 2017.
authors.affiliation: Early Stage Cell Culture, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Lam
authors.affiliation: Early Discovery Biochemistry, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Lydia
authors.initials: L
authors.lastname: Santell
authors.affiliation: Biochemical and Cellular Pharmacology Dept., Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Blair
authors.initials: B
authors.lastname: Wilson
authors.affiliation: Early Stage Cell Culture, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Mandy
authors.initials: M
authors.lastname: Yim
authors.affiliation: Early Stage Cell Culture, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Salina
authors.initials: S
authors.lastname: Louie
authors.affiliation: Early Stage Cell Culture, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Danming
authors.initials: D
authors.lastname: Tang
authors.affiliation: Early Stage Cell Culture, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: David
authors.initials: D
authors.lastname: Shaw
authors.affiliation: Biochemical and Cellular Pharmacology Dept., Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Pamela
authors.initials: P
authors.lastname: Chan
authors.affiliation: Early Discovery Biochemistry, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lazarus
authors.affiliation: Early Stage Cell Culture, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Brad
authors.initials: B
authors.lastname: Snedecor
authors.affiliation: Early Stage Cell Culture, Genentech, Inc, South San Francisco, CA, 94080.
authors.forename: Shahram
authors.initials: S
authors.lastname: Misaghi
mesh_terms: D000199:Actins / Q000737:chemistry*; D000818:Animals; D016466:CHO Cells; D049109:Cell Proliferation / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods*; D003412:Cricetulus; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012492:Salts / Q000737:chemistry*
chemical_list: D000199:Actins; D011994:Recombinant Proteins; D012492:Salts; C107519:DNASE1L2 protein, human; D003850:Deoxyribonuclease I
keywords: cystic fibrosis; hDNase I; hyperactive; inducible expression