Fig. 1: Expression of human AAT in murine MΦ.

A Design of the third-generation, self-inactivating lentiviral vector expressing human M-type α1 antitrypsin (AAT) cDNA coupled to an enhanced green fluorescence protein (eGFP) reporter by an internal ribosomal entry site (IRES) from the Cbx-EFS, CAG, or Cbx-EF1α promoter, respectively (left). Schematic design of the control vector expressing the eGFP after an IRES sequence under the Cbx-EFS promoter (right). 5’ LTR 5’ long terminal repeat, ψ packaging signal, wPRE Woodchuck hepatitis virus posttranscriptional regulatory element, 3’ ΔLTR 3’ long terminal repeat with deletion leading to self-inactivation; EFS, EF1α: elongation factor 1α short (EFS) or long version (EF1α); Cbx: element derived from the 5’ part of the HNRPA2B1/CBX3 ubiquitous chromatin opening element, CAG: synthetic promoter composed of the cytomegalovirus early enhancer, the chicken beta-actin promoter and the splice acceptor of the rabbit beta-globin gene. B Scheme of the experimental procedure. Lineage negative (lin^-) cells were isolated from bone marrow of wildtype (WT) C57BL/6J mice, transduced with AAT or eGFP control vectors, sorted for eGFP expression, differentiated into macrophages (MΦ), and subsequently used for evaluation of MΦ functionality and AAT expression as well as AAT functionality. Created with BioRender.com. C Percentage of eGFP⁺ cells before sorting representing the efficiency of lin^- cell transduction of the lentiviral vectors. D Determination of the vector copy number (VCN) per genome after lentiviral transduction and eGFP⁺ sorting in murine MΦ. E Representative histograms of the eGFP expression after lentiviral transduction and eGFP⁺ sorting in murine MΦ. F Median fluorescence intensity (MFI) of eGFP after lentiviral transduction and eGFP⁺ sorting in murine MΦ. eGFP n = 3; mock n = 4; Cbx-EFS-AAT, CAG-AAT, Cbx-EF1α-AAT n = 5. G Expression of human SERPINA1 mRNA after lentiviral transduction and eGFP⁺ sorting in murine MΦ measured by RT-qPCR. H Representative human AAT western blot analysis of MΦ supernatant and MΦ lysates. Human serum was used as a positive control. Vinculin band in cellular lysates serves as a loading control. I ELISA quantification of human AAT secretion by murine MΦ. Asterisks mark the samples that were used for the representative western blot in (H). Each point in graphs and all n-numbers given represent independent biological experiments, which is an individual isolation of lin^- cells and individual transduction. Lines and error bars represent mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.