Gene therapy for pulmonary arterial hypertension with bone morphogenetic protein receptor type-2 modulation via engineered endothelial progenitor cells or a targeted adeno-viral construct: changes in Smad and non-Smad signalling contributed to amelioration of disease

Abstract

Pulmonary arterial hypertension (PAH) is a rare but devastating disease and despite available therapeutics, survival remains at 3-5 years. Reduced expression of the bone morphogenetic protein receptor type 2 (BMPR2) is causally linked to hereditary, idiopathic and secondary forms of PAH. Thus, we proposed that up-regulation of BMPR2 may be therapeutic. As proof of concept, we’ve previously attenuated PAH in animal models through BMPR2 targeted gene delivery using Adenoviral (Ad) vectors. However, further understanding of the cell signalling mechanisms involved, as well as overcoming limitations with viral vector approaches is required to progress this approach to the clinic. Endothelial progenitor cells (EPCs) may be the key to avoiding the shortcomings of Ad-vector technology. EPCs are important for angiogenesis as well as tissue repair and have been shown to have altered function and abundance in patients with PAH. Manipulating these cells may be an alternate means to up-regulate BMPR2 in lungs affected by PAH, thereby avoiding some of the limitations of viral gene delivery techniques and enabling easier clinical translation. Herein, I confirmed disease reversal in the rat monocrotaline (MCT)- induced PAH model following targeted gene delivery of BMPR2 to the pulmonary vascular endothelium and assessed the relevant BMPR2 mediated Smad pathways in whole lung tissue, 10 days following treatment. Microarray technology was utilised to identify any novel molecular targets, with results from this indicating that a peak Smad signalling effect was missed at this 10 day time-point. However, the microarray did indicate potential changes in BMPR2 mediated non-Smad signalling. PAH reversal was then assessed 2 days following targeted gene delivery of BMPR2 to the pulivmonary endothelium and further assessment of BMPR2 mediated Smad and non-Smad pathways were analysed in the subsequent whole lung tissue. Moving towards a more clinically applicable therapy, cell therapy using ex vivo engineered EPCs to deliver BMPR2 to the pulmonary endothelium was investigated in the rat MCT-induced PAH model. To do this, the technique to isolate and culture rat bone marrow derived EPCs (r-EPCs) was developed. Successful transduction of these cells to over-express BMPR2 was optimised and these now engineered cells were used as a vehicle to deliver BMPR2 to the pulmonary vasculature via intravenous injection into rats with MCT-induced PAH. Amelioration of PAH was confirmed 10 days following the cell therapy treatment and subsequent protein analysis of BMPR2 mediated Smad pathways in the whole lung tissue saw changes activated Smad1/5/8. The development of new therapies for PAH is critical. BMPR2 modulation is a novel therapeutic strategy which addresses the well known underlying pathology of BMPR2 deficiency that occurs not only in hereditary PH, but secondary PH and most PAH animal models. The success of our highly novel pre-clinical BMPR2 cell therapy may lead the way for further development of other BMPR2 therapies, as well as give significant insight into the pathophysiology of this devastating disease.