HUMAN GENE THERAPY 11:403–414 (February 10, 2000) Mary Ann Liebert, Inc. Engineering of a Glucose-Responsive Surrogate Cell for Insulin Replacement Therapy of Experimental Insulin-Dependent Diabetes MARKUS TIEDGE, 1 MATTHIAS ELSNER, 1 NEVILLE HUGO MC CLENAGHAN, 1 HANS-JÜRGEN HEDRICH, 2 DIETRICH GRUBE, 3 JÜRGEN KLEMPNAUER 4 and SIGURD LENZEN 1 ABSTRACT Glucose responsiveness in the millimolar concentration range is a crucial requirement of a surrogate pan- creatic beta cell for insulin replacement therapy of insulin-dependent diabetes. Novel insulin-secreting GK cell clones with millimolar glucose responsiveness were generated from an early-passage glucose-unrespon- sive RINm5F cell line. This line expressed constitutively both the K ATP channel and the GLUT2 glucose trans- porter; but it had a relative lack of glucokinase. Through overexpression of glucokinase, however, it was pos- sible to generate glucose-responsive clones with a glucokinase-to-hexokinase ratio comparable to that of a normal pancreatic beta cell. This aim, on the other hand, was not achieved through overexpression of the GLUT2 glucose transporter. Raising the expression level of this glucose transporter into the range of rat liver, without correcting the glucokinase-to-hexokinase enzyme ratio, did not render the cells glucose responsive. These glucokinase-overexpressing RINm5F cells also stably maintained their molecular and insulin secretory characteristics in vivo. After implantation into streptozotocin diabetic immunodeficient rats, glucokinase-over- expressing cells retained their insulin responsiveness to physiological glucose stimulation under in vivo con- ditions. These cells represent a notable step toward the future bioengineering of a surrogate beta cell for in- sulin replacement therapy in insulin-dependent diabetes mellitus. 403 OVERVIEW SUMMARY Physiological glucose responsiveness is the hallmark of a surrogate pancreatic beta cell for insulin replacement ther- apy in insulin-dependent diabetes. In this study millimolar glucose responsiveness of insulin secretion was achieved in RINm5F insulin-producing tissue culture cells through overexpression of the glucose-sensin g enzyme glucokinase. The bioengineered RINm5F-GK cells showed molecular and metabolic characteristics comparable to those of the normal pancreatic beta cell, with a low basal level of insulin release and a 2.6-fold increase in insulin secretion at 5 mM glucose. Implantation of glucose-responsive surrogate RINm5F-GK cells normalized the hyperglycemic metabolic state of streptozotocin-diabetic rats with a stable expression level of the glucokinase transgene. After an oral glucose load implanted RINm5F-GK cells induced a significant physio- logical increase of serum insulin. This study demonstrates conclusively the in vivo suitability of bioengineered glucose- responsive insulin-producing surrogate cells for insulin re- placement therapy in IDDM. INTRODUCTION B IOENGINEERING of insulin-producing cells is a potential strategy to overcome the limited availability of human pan- creatic donor tissue for insulin replacement therapy in diabetic patients. Two different approaches have been taken to bio- engineer insulin-producing cells that may be used as surrogate beta cells. The expression of insulin in nonendocrine cells, e.g., fibroblasts, myoblasts, or hepatocytes, has gained a successful experimental basis as demonstrated by the ability of these in- sulin-releasing cells to decrease hyperglycemia in diabetic an- imals (Simonson et al., 1996; Gros et al. , 1997, 1999; Falqui et al. , 1999a,b). Through genetic modification of biopsied cells 1 Institute of Clinical Biochemistry, 2 Institute for Laboratory Animal Science, 3 Institute of Anatomy I, and 4 Department of Surgery, Hannover Medical School, D-30623 Hannover, Germany.