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We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

[This corrects the article on p. e50630 in vol. 7.].

OBJECTIVES: Glucagon-like peptide 2 (GLP2) is an intestinal growth factor that has been shown to stimulate intestinal growth and reduce disease severity in preclinical models of short bowel syndrome and inflammatory bowel disease. Teduglutide, a recombinant human GLP2 variant (GLP2-2G), has increased half-life and stability as compared to the native GLP2 peptide, but still requires twice daily dosing in preclinical models and daily dosing in the clinic. The goal of this study was to produce and characterize the preclinical pharmacokinetic and therapeutic properties of GLP2-2G-XTEN, a novel, long-acting form of GLP2-2G. METHODOLOGY AND RESULTS: A GLP2-2G-XTEN fusion protein with extended exposure profile was produced by genetic fusion of GLP2-2G peptide to XTEN, a long, unstructured, non-repetitive, hydrophilic sequence of amino acids. The serum half-life of GLP2-2G-XTEN in mice, rats and monkeys was 34, 38 and 120 hours, respectively. Intestinotrophic effects were demonstrated in normal rats, where GLP2-2G-XTEN administration resulted in a significant increase in both small intestine weight and length. Efficacy of the GLP2-2G-XTEN protein was compared to that of GLP2-2G peptide in a rat Crohn's disease model, indomethacin-induced inflammation. Prophylactic administration of GLP2-2G-XTEN significantly increased the length, reduced the number of trans-ulcerations and adhesions, and reduced the TNFα content of the small intestine. GLP2-2G-XTEN demonstrated greater in vivo potency as compared to GLP2-2G peptide, and improvement in histopathology supported the GLP2-2G-XTEN treatment effects. CONCLUSIONS AND SIGNIFICANCE: GLP2-2G-XTEN is intestinotrophic and demonstrates efficacy in a rat Crohn's disease model requiring a lower molar dose and less frequent dosing relative to GLP2-2G peptide. Allometric scaling based on pharmacokinetics from mouse, rat and monkey projects a human half-life of 240 hours. These improvements in preclinical pharmacokinetics and dosing indicate that GLP2-2G-XTEN may offer a superior therapeutic benefit for treatment of gastrointestinal diseases including Crohn's disease.

While the majority of current diabetes treatments focus on reducing blood glucose levels, hypoglycemia represents a significant risk associated with insulin treatment. Glucagon plays a major regulatory role in controlling hypoglycemia in vivo, but its short half-life and hyperglycemic effects prevent its therapeutic use for non-acute applications. The goal of this study was to identify a modified form of glucagon suitable for prophylactic treatment of hypoglycemia without increasing baseline blood glucose levels.Through application of the XTEN technology, we report the construction of a glucagon fusion protein with an extended exposure profile (Gcg-XTEN). The in vivo half-life of the construct was tuned to support nightly dosing through design and testing in cynomolgus monkeys. Efficacy of the construct was assessed in beagle dogs using an insulin challenge to induce hypoglycemia. Dose ranging of Gcg-XTEN in fasted beagle dogs demonstrated that the compound was biologically active with a pharmacodynamic profile consistent with the designed half-life. Prophylactic administration of 0.6 nmol/kg Gcg-XTEN to dogs conferred resistance to a hypoglycemic challenge at 6 hours post-dose without affecting baseline blood glucose levels. Consistent with the designed pharmacokinetic profile, hypoglycemia resistance was not observed at 12 hours post-dose. Importantly, the solubility and stability of the glucagon peptide were also significantly improved by fusion to XTEN.The data show that Gcg-XTEN is effective in preventing hypoglycemia without the associated hyperglycemia expected for unmodified glucagon. While the plasma clearance of this Gcg-XTEN has been optimized for overnight dosing, specifically for the treatment of nocturnal hypoglycemia, constructs with significantly longer exposure profiles are feasible. Such constructs may have multiple applications such as allowing for more aggressive insulin treatment regimens, treating hypoglycemia due to insulin-secreting tumors, providing synergistic efficacy in combination therapies with long-acting GLP1 analogs, and as an appetite suppressant for treatment of obesity. The improved physical properties of the Gcg-XTEN molecule may also allow for novel delivery systems not currently possible with native glucagon.