RESUMEN
Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings.
Asunto(s)
Lentivirus/genética , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Ratones , FN-kappa B/genética , Interferencia de ARN/fisiología , Factor de Transcripción ReIA/genéticaRESUMEN
Two populations of Nkx2-1(+) progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFß and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1(GFP) knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.
Asunto(s)
Separación Celular , Células Madre Embrionarias/citología , Pulmón/citología , Glándula Tiroides/citología , Animales , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Células Madre Embrionarias/metabolismo , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Ratones , Ratones Transgénicos , Transducción de Señal/fisiología , Glándula Tiroides/embriología , Glándula Tiroides/metabolismo , Andamios del TejidoRESUMEN
Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human alpha1 antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.
Asunto(s)
Enfisema/terapia , Terapia Genética , Lentivirus/genética , Macrófagos Alveolares/metabolismo , alfa 1-Antitripsina/genética , Animales , Humanos , Inflamación/etiología , Antígenos Comunes de Leucocito/análisis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Transducción Genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
Inherited mutations in the human alpha(1)-antitrypsin (AAT) gene lead to deficient circulating levels of AAT protein and a predisposition to developing emphysema. Gene therapy for individuals deficient in AAT is an attractive goal, because transfer of a normal AAT gene into any cell type able to secrete AAT should reverse deficient AAT levels and attenuate progression of lung disease. Here we present an approach for AAT gene transfer based on the transplantation of lentivirally transduced hematopoietic stem cells (HSCs). We develop a novel dual-promoter lentiviral system to transfer normal human AAT cDNA as well as a fluorescent tracking "reporter gene" into murine HSCs. After transplantation of 3,000 transduced HSCs into irradiated mouse recipients, we demonstrate simultaneous and sustained systemic expression of both genes in vivo for at least 31 weeks. The stem cells transduced with this protocol maintain multipotency, self-renewal potential, and the ability to reconstitute the hematopoietic systems of both primary and secondary recipients. This lentiviral-based system may be useful for investigations requiring the systemic secretion of anti-proteases or cytokines relevant to the pathogenesis of a variety of lung diseases.