Modification of gene expression and increase in α1-Antitrypsin (α1-AT) secretion after homologous recombination in α1-AT-Deficient monocytes

Small DNA fragments (SDFs) including normal M and alpha(1)-antitrypsin deficiency (alpha(1)-ATD) Z sequences were generated and transfected into peripheral blood monocytes from M subjects and Z alpha(1)-ATD patients. Untreated M and alpha(1)-ATD monocytes secreted 32 +/- 1.1 and 23 +/- 1.4 ng of alpha(1)-AT per 10(6) monocytes over 24 hr. After tumor necrosis factor (TNF)-alpha stimulation, the alpha(1)-AT secretion from M monocytes increased significantly to 50 +/- 2.1 ng/10(6) over 24 hr (p = 0.0004), whereas there was no change in secreted alpha(1)-AT from TNF-alpha-stimulated alpha(1)-ATD monocytes. However, after Z SDF transfection, M monocytes failed to increase alpha(1)-AT secretion in response to TNF-alpha stimulation. Transfecting alpha(1)-ATD monocytes with the M SDF resulted in a significant increase in alpha(1)-AT secretion (p = 0.03) after TNF-alpha stimulation to 55 +/- 2.7 ng/106 cells. Monocytes from a further 13 alpha(1)-ATD patients constitutively produced alpha(1)-AT after the first 24 hr. Transfection with either transfection reagent alone or with Z SDF slightly increased alpha(1)-AT secretion over the subsequent 24 hr. However, M SDF transfection significantly increased alpha(1)-AT secretion further, compared with untreated or sham transfection. Untreated, transfection reagent-treated, and Z SDF-transfected alpha(1)-ATD monocytes generated polymerase chain reaction products from Z primers. M SDF-treated alpha(1)-ATD monocytes generated bands with M primers, indicating the generation of a corrected transcript. In conclusion, the defective gene can be corrected in alpha(1)-ATD monocytes with SDFs, and treatment is associated with an increase in alpha(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.