HUMAN GENE THERAPY 18:1171–1177 (November 2007) © Mary Ann Liebert, Inc. DOI: 10.1089/hum.2007.073 Modification of Gene Expression and Increase in  1 -Antitrypsin ( 1 -AT) Secretion After Homologous Recombination in  1 -AT-Deficient Monocytes GILLIAN L. MCNAB, ALI AHMAD, DIPPICA MISTRY, and ROBERT A. STOCKLEY ABSTRACT Small DNA fragments (SDFs) including normal M and  1 -antitrypsin deficiency ( 1 -ATD) Z sequences were generated and transfected into peripheral blood monocytes from M subjects and Z  1 -ATD patients. Un- treated M and  1 -ATD monocytes secreted 32  1.1 and 23  1.4 ng of  1 -AT per 10 6 monocytes over 24 hr. After tumor necrosis factor (TNF)- stimulation, the  1 -AT secretion from M monocytes increased signifi- cantly to 50  2.1 ng/10 6 over 24 hr (p  0.0004), whereas there was no change in secreted  1 -AT from TNF- -stimulated  1 -ATD monocytes. However, after Z SDF transfection, M monocytes failed to increase  1 -AT secretion in response to TNF- stimulation. Transfecting  1 -ATD monocytes with the M SDF resulted in a significant increase in  1 -AT secretion (p  0.03) after TNF- stimulation to 55  2.7 ng/10 6 cells. Monocytes from a further 13  1 -ATD patients constitutively produced  1 -AT after the first 24 hr. Transfection with ei- ther transfection reagent alone or with Z SDF slightly increased  1 -AT secretion over the subsequent 24 hr. However, M SDF transfection significantly increased  1 -AT secretion further, compared with untreated or sham transfection. Untreated, transfection reagent-treated, and Z SDF-transfected  1 -ATD monocytes gen- erated polymerase chain reaction products from Z primers. M SDF-treated  1 -ATD monocytes generated bands with M primers, indicating the generation of a corrected transcript. In conclusion, the defective gene can be corrected in  1 -ATD monocytes with SDFs, and treatment is associated with an increase in  1 -AT se- cretion. 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. 1171 INTRODUCTION T HE 54-kDa serum glycoprotein  1 -antitrypsin ( 1 -AT) is encoded by a gene located on chromosome 14 at posi- tion q32.1. It is produced mainly in the liver and also by alve- olar macrophages and peripheral blood monocytes.  1 -AT is a polymorphic protein with more than 120 variants, most associ- ated with normal or slightly reduced  1 -AT levels. The com- monest severe form of  1 -antitrypsin deficiency ( 1 -ATD) is associated with the PiZ variant, which is caused by a point mu- tation at position 342 in exon V of the  1 -AT gene. This causes an amino acid substitution from glutamic acid to lysine (Crys- tal, 1990). This base change distorts the reactive central loop and -sheet A within the  1 -AT molecule, leading to the for- mation of polymers that accumulate within the endoplasmic reticulum of hepatocytes. The accumulation of  1 -AT in the liver leads to a decrease in serum concentrations to 10–15% normal and an increased risk of developing liver disease. Cir- rhosis results from cytotoxicity associated with accumulation of mutant  1 -AT in the hepatocytes, although the exact mech- anism remains unknown. A low concentration of  1 -AT in serum and the lung can predispose to lung disease, as there is insufficient  1 -AT to prevent serine proteinases, particularly neutrophil elastase, from causing lung damage by destruction of lung tissue, thus leading to the development of emphysema (Stockley, 2000).  1 -Antitrypsin deficiency has been treated with intravenous augmentation therapy, which increases serum  1 -AT to levels that should protect the lungs. Alternative treatments to increase  1 -AT levels include  1 -AT gene transfer, using a variety of Department of Respiratory Medicine, University of Birmingham, Birmingham B15 2TT, United Kingdom.