Johannes Geiger

Targeting of the β2-adrenoceptor increases nonviral gene delivery to pulmonary epithelial cells in vitro and lungs in vivo

Coupling of targeting ligands to polyethylenimine (PEI) has been previously used to improve transfection efficiency of PEI gene vectors. Here, we show that the beta(2)-adrenoceptor (beta(2)-AR) agonist, clenbuterol (Clen), can be used to improve gene transfer efficiency of PEI gene vectors on alveolar epithelial cells in vitro and in the lungs of mice in vivo. Clenbuterol conjugated to fluorescein-labeled bovine serum albumin resulted in clenbuterol-specific cellular uptake predominantly into alveolar but not bronchial epithelial cells. Clen-g-PEI (4/1) conjugates were combined with increasing molar ratios of PEI for transfection. At optimized PEI-g-Clen/PEI composition, transfection efficiency on alveolar epithelial cells was up to 14-fold higher than for unmodified PEI and could be inhibited by an excess of free clenbuterol. No increase of transfection efficiency was observed on bronchial epithelial cells. Increasing the PEI-g-Clen/PEI molar ratio resulted in an increase of gene vector size, decrease of the zeta potential and cytotoxicity. Aerosol delivery of optimized PEI-g-Clen/PEI (1/5) gene vectors resulted in a significant 3-fold increase of gene expression in the lungs of mice compared with unmodified PEI gene vectors. We suggest that coupling of beta(2)-adrenoceptor ligands to nonviral gene vectors represents a promising approach to improve gene delivery to the lungs.

Vectors for pulmonary gene therapy

The success of gene transfer in preclinical animal models and proof of principle clinical studies has made gene therapy an attractive concept for disease treatment. A variety of diseases affecting the lung are candidates for gene therapy. Delivery of genes to the lungs seems to be straightforward, because of the easy accessibility of epithelial cells via the airways. However, efficient delivery and expression of the therapeutic transgene at levels sufficient to result in phenotypic correction of the diseased state have proven elusive. This review presents a brief summary about current status and future prospects in the development of viral and non-viral strategies for pulmonary gene therapy.

Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues.

Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2-4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (F(ILO) (ILO:PEI) = 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, F(ILO) = 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO F(ILO) = 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer.

Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA

Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models.

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.