Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase-mediated cassette exchange system.

Cell line development (CLD) by random integration (RI) can be labor intensive, inconsistent, and unpredictable due to uncontrolled gene integration after transfection. Unlike RI, targeted integration (TI) based CLD introduces the antibody-expressing cassette to a predetermined site by recombinase-mediated cassette exchange (RMCE). The key to success for the development of a TI host for therapeutic antibody production is to identify a transcriptionally active hotspot that enables highly efficient RMCE and antibody expression with good stability. In this study, a genome wide search for hotspots in the Chinese hamster ovary (CHO)-K1-M genome by either RI or PiggyBac (PB) transposase-based integration has been described. Two CHO-K1-M derived TI host cells were established with the Cre/Lox RMCE system and are described here. Both TI hosts contain a GFP-expressing landing pad flanked by two incompatible LoxP recombination sites (L3 and 2L). In addition, a third incompatible LoxP site (LoxFAS) is inserted in the GFP landing pad to enable an innovative two-plasmid based RMCE strategy, in which two separate vectors can be targeted to a single locus simultaneously. Cell lines generated by the TI system exhibit comparable or higher productivity, better stability and fewer sequence variant (SV) occurrences than the RI cell lines.

Concurrent transfection of randomized transgene configurations into targeted integration CHO host is an advantageous and cost-effective method for expression of complex molecules.

Complex recombinant proteins are increasingly desired as potential therapeutic options for many disease indications and are commonly expressed in the mammalian Chinese hamster ovary (CHO) cells. Generally, stoichiometric expression and proper folding of all subunits of a complex recombinant protein are required to achieve the desired titers and product qualities for a complex molecule. Targeted integration (TI) cell line development (CLD), which entails the insertion of the desired transgene(s) into a predefined landing-pad in the CHO genome, enables the generation of a homogeneous pool of cells from which clonally stable and high titer clones can be isolated with minimal screening efforts. Despite these advantages, using a single transgene(s) configuration with predetermined gene dosage might not be adequate for the expression of complex molecules. The goal of this study is to develop a method for seamless screening of many vector configurations in a single TI CLD attempt. As testing vector configurations in transient expression systems is not predictive of protein expression in the stable cell lines and parallel TI CLDs with different transgene configurations is resource-intensive, we tested the concept of randomized configuration targeted integration (RCTI) CLD approach for expression of complex molecules. RCTI allows simultaneous transfection of multiple vector configurations, encoding a complex molecule, to generate diverse TI clones each with a single transgene configuration but clone specific productivity and product qualities. Our findings further revealed a direct correlation between transgenes' configuration/copy-number and titer/product quality of the expressed proteins. RCTI CLD enabled, with significantly fewer resources, seamless isolation of clones with comparable titers and product quality attributes to that of several parallel standard TI CLDs. Therefore, RCTI introduces randomness to the TI CLD platform while maintaining all the advantages, such as clone stability and reduced sequence variant levels, that the TI system has to offer.

Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3.

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell.

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass.

Cloning, expression and functional characterization of cytochrome P450 3A37 from turkey liver with high aflatoxin B1 epoxidation activity.

Cytochrome P450s (P450) play an important role in the formation of carcinogenic and mutagenic electrophilic intermediates from a wide range of xenobiotics, including naturally occurring dietary compounds. The pathogenesis of hepatotoxic and hepatocarcinogenic action of the mycotoxin aflatoxin B(1) (AFB(1)) involves initial bioactivation by P450s to a reactive and electrophilic intermediate exo-aflatoxin B(1)-8,9-epoxide (exo-AFBO). Poultry, especially turkeys are extremely sensitive to AFB(1), a condition due, in part, to efficient epoxidation by P450 1A and 3A enzymes. We previously reported the discovery of P450 1A5 from turkey liver, which like its human homologue, 1A2, bioactivated AFB(1) to exo-AFBO and aflatoxin M(1) (AFM(1)). Here, we describe P450 3A37, the 3A4 homologue from turkey liver. This gene has an open reading frame (ORF) of 1512 bp, and the protein is predicted to be 504 amino acids with 97% identity to chicken P450 3A37. A truncated construct of the turkey P450 3A37 gene with 11 amino acids deleted from the hydrophobic N-terminal region was heterologously expressed in Escherichia coli, the protein from which exhibited a CO difference spectrum typical of P450s. Like human P450 3A4, 3A37 biotransformed AFB(1) to exo-AFBO and aflatoxin Q(1) (AFQ(1)) and possessed nifedipine oxidation activity, both of which were inhibited by the P450 3A4 inhibitor 17alpha-ethynylestradiol. Oxidation of AFB(1) to exo-AFBO and AFQ(1) by P450 3A37 followed sigmoidal Hill kinetics, suggestive of an allosteric interaction between the enzyme and AFB(1). The Hill coefficient (n) value was 1.9 for exo-AFBO and 1.6 for AFQ(1), indicative of positive cooperativity. The calculated K(m) and V(max) values for the formation of exo-AFBO were 287 +/- 21 muM and 1.45 +/- 0.07 nmol/min/nmol P450, respectively, whereas those of AFQ(1) formation were 302 +/- 51 muM and 7.86 +/- 0.75 nmol/min/nmol P450, respectively. These data strongly suggest that P450 3A37, along with P450 1A5, plays an important role in AFB(1) epoxidation in turkey liver.

Molecular cloning and expression of a novel cytochrome p450 from turkey liver with aflatoxin b1 oxidizing activity.

Cytochromes P450 are members of a superfamily of oxidative hemoprotein enzymes that metabolize a variety of endogenous and exogenous compounds. Previous studies in our laboratory have shown that efficient P450-mediated activation underlies the extreme sensitivity of poultry, specifically turkeys, to the toxic effects of the mycotoxin aflatoxin B1 (AFB1). Using 3'- and 5'-rapid amplification of cDNA ends (RACE), we amplified from turkey liver RNA a full-length 1.73 kb cDNA predicted to be 528 amino acids with 94.7% sequence identity to a CYP1A5 from chicken liver. A truncated construct of the turkey CYP1A5 gene with 29 amino acids deleted from the hydrophobic NH2-terminal region was cloned and heterologously expressed in Escherichia coli. The expressed protein from E. coli membranes had a CO-binding spectrum typical of P450s, and it catalyzed the O-dealkylation of the CYP1A prototype substrates ethoxyresorufin and methoxyresorufin. CYP1A5-mediated O-dealkylation of methoxyresorufin was completely inhibited by alpha-naphthoflavone, a specific CYP1A inhibitor. Inhibitors to other mammalian P450s (3A4, 2D, 2E, and 3A1) either slightly inhibited this activity or not at all. CYP1A5 oxidized AFB1 to form two metabolites: the reactive intermediate, AFB1 -8,9-epoxide (AFBO), and aflatoxin M1 (AFM1). Because of the importance of AFBO and AFM1 in the toxicity of AFB1, we conclude that this P450 probably plays some role in the well-known hypersensitivity of turkeys to AFB1. To our knowledge, this is the first P450 cloned and sequenced from turkeys, the species in which the toxicity of AFB1 was first discovered.