Modification of gene expression and increase in alpha1-antitrypsin (alpha1-AT) secretion after homologous recombination in alpha1-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/10(6) 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.
McNab
Gillian L
GL
Department of Respiratory Medicine, University of Birmingham, Birmingham B15 2TT, United Kingdom. G.L.McNab.1@bham.ac.uk
Ahmad
Ali
A
Mistry
Dippica
D
Stockley
Robert A
RA
D001483:Base Sequence; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005838:Genotype; D006720:Homozygote; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D017385:Sequence Homology; D014162:Transfection; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism*; D019896:alpha 1-Antitrypsin Deficiency / Q000235:genetics*
D000515:alpha 1-Antitrypsin