Gene Therapy Center; Department of Pediatrics
Terence R. Flotte
Medical Subject Headings
alpha 1-Antitrypsin Deficiency; alpha 1-Antitrypsin; Gene Therapy; Codon
Congenital, Hereditary, and Neonatal Diseases and Abnormalities | Genetic Processes | Genetics and Genomics | Life Sciences | Medicine and Health Sciences | Respiratory Tract Diseases
Alpha 1-antitrypsin deficiency is a genetic disorder caused by defective production of alpha 1-antitrypsin (AAT). Gene therapy approaches have been conducted in patients with AAT deficiency with successful AAT expression, but not to the therapeutic levels required to reduce the risk of emphysema. Codon optimization, a somewhat new and evolving technique, is used by many scientists to maximize protein expression in living organisms by altering translational and transcriptional efficiency as well as protein refolding. The purpose of this study was to develop single stranded and double stranded AAT gene constructs, test their protein expression in vitro, and compare with those levels expressed by the AAT construct that is currently in clinical trials. Three constructs were to be developed, yet only one construct was successfully cloned. This clone, optimized ds-CB-AAT, illustrated increased AAT protein expression as the transfection time increased. However, protein levels were appreciably lower in the optimized construct compared to the single stranded (long intron) AAT construct that is currently being administered in clinical trials. The data did not suggest that the optimized AAT construct does in fact express more AAT protein in vitro as expected. In order to achieve data that can be reproduced, the 2 remaining constructs need to be cloned and all of the isolated plasmid DNA should be prepared on the same scale to minimize any additional confounding variables.
Menz, Timothy; Tang, Qiushi; Song, Lina; Mueller, Christian; and Flotte, Terence R., "Codon Optimization for Alpha 1-Antitrypsin Disease" (2012). University of Massachusetts Medical School. Senior Scholars Program. Paper 122.