Modification of Gene Expression and Increase in α 1 -Antitrypsin ( α 1 -AT) Secretion After Homologous Recombination in α 1 -AT-Deficient Monocytes

Small DNA fragments (SDFs) including normal M and α ₁-antitrypsin deficiency (α ₁-ATD) Z sequences were generated and transfected into peripheral blood monocytes from M subjects and Z α ₁-ATD patients. Untreated M and α ₁-ATD monocytes secreted 32 ± 1.1 and 23 ± 1.4 ng of α ₁-AT per 10⁶ monocytes over 24 hr. After tumor necrosis factor (TNF)-α stimulation, the α ₁-AT secretion from M monocytes increased significantly to 50 ± 2.1 ng/10⁶ over 24 hr (p = 0.0004), whereas there was no change in secreted α ₁-AT from TNF-α-stimulated α ₁-ATD monocytes. However, after Z SDF transfection, M monocytes failed to increase α ₁-AT secretion in response to TNF-α stimulation. Transfecting α ₁-ATD monocytes with the M SDF resulted in a significant increase in α ₁-AT secretion (p = 0.03) after TNF-α stimulation to 55 ± 2.7 ng/10⁶ cells. Monocytes from a further 13 α ₁-ATD patients constitutively produced α ₁-AT after the first 24 hr. Transfection with either transfection reagent alone or with Z SDF slightly increased α ₁-AT secretion over the subsequent 24 hr. However, M SDF transfection significantly increased α ₁-AT secretion further, compared with untreated or sham transfection. Untreated, transfection reagent-treated, and Z SDF-transfected α ₁-ATD monocytes generated polymerase chain reaction products from Z primers. M SDF-treated α ₁-ATD monocytes generated bands with M primers, indicating the generation of a corrected transcript. In conclusion, the defective gene can be corrected in α ₁-ATD monocytes with SDFs, and treatment is associated with an increase in α ₁-AT secretion. The development of this methodology to repair the gene defect in hepatocytes should have beneficial effects on secretion, thereby protecting both the lung and liver.