Pulmonary transplantation of alpha-1 antitrypsin (AAT)-transgenic macrophages provides a source of functional human AAT in vivo

Ewa Janosz,Axel Schambach,Hanna Spielmann,Charlotte Rossdam,Miriam Hetzel,Sabina Janciauskiene,Thomas Moritz,Nico Lachmann,Marc Schwabbauer,Constantin Von Kaisenberg,Srinu Tumpara,Doreen Kloos,Falk F R Buettner

doi:10.1038/s41434-021-00269-3

Ewa Janosz, Axel Schambach + Show 11 more

Open Access

https://doi.org/10.1038/s41434-021-00269-3

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Abstract

Inherited deficiency of the antiprotease alpha-1 antitrypsin (AAT) is associated with liver failure and early-onset emphysema. In mice, in vivo lentiviral transduction of alveolar macrophages (AMs) has been described to yield protective pulmonary AAT levels and ameliorate emphysema development. We here investigated the pulmonary transplantation of macrophages (PMT) transgenic for AAT as a potential therapy for AAT deficiency-associated lung pathology. Employing third-generation SIN-lentiviral vectors expressing the human AAT cDNA from the CAG or Cbx-EF1α promoter, we obtained high-level AAT secretion in a murine AM cell line as well as murine bone marrow-derived macrophages differentiated in vitro (AAT MΦ). Secreted AAT demonstrated a physiologic glycosylation pattern as well as elastase-inhibitory and anti-apoptotic properties. AAT MΦ preserved normal morphology, surface phenotype, and functionality. Furthermore, in vitro generated murine AAT MΦ successfully engrafted in AM-deficient Csf2rb–/– mice and converted into a CD11c+/Siglec-F+ AM phenotype as detected in bronchoalveolar lavage fluid and homogenized lung tissue 2 months after PMT. Moreover, human AAT was detected in the lung epithelial lining fluid of transplanted animals. Efficient AAT expression and secretion were also demonstrated for human AAT MΦ, confirming the applicability of our vectors in human cells.

Highlights

Alpha-1 antitrypsin (AAT) is an acute phase glycoprotein produced and secreted into the serum predominantly by hepatocytes and to a lesser extent by other cells, including intestinal epithelium, neutrophils, and macrophages (MΦ) [1–3]
For stable transgene overexpression in MΦ, we employed third-generation SIN lentiviral vectors (LVs) expressing the human healthy “M” type alpha-1 antitrypsin (AAT) cDNA coupled to an eGFP reporter by an internal ribosomal entry site (IRES)
Three different promoter constructs were evaluated: elongation factor 1α short (EFS) or long version (EF1α) both coupled with the CBX3 ubiquitous chromatin opening element shown to improve expression levels and reduce transgene silencing [30] and the synthetic CAG promoter composed of the cytomegalovirus early enhancer, the chicken beta-actin promoter and the splice acceptor of the rabbit beta-globin gene (CAG-AAT) [31]

Summary

Introduction

Alpha-1 antitrypsin (AAT) is an acute phase glycoprotein produced and secreted into the serum predominantly by hepatocytes and to a lesser extent by other cells, including intestinal epithelium, neutrophils, and macrophages (MΦ) [1–3]. The only therapy available for AATDrelated lung disease is substitution therapy employing weekly intravenous infusion of AAT purified from human plasma [12, 13]. This augmentation therapy is associated with high costs, limited availability, and the risk of transmitting pathogens. In this context, a variety of genetic approaches aiming to provide constant production of transgenic AAT have been pursued. Other studies investigated intrapulmonary delivery of AAT-encoding lentiviral vectors (LVs) and demonstrated amelioration of elastase-induced emphysema in mice after transduction of alveolar macrophages (AMs) [20], or lung epithelial cells [21] depending on the pseudotype of the vector

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