(591) Modulation of the DNP Interactome Enhances CFTR Gene Transfer in a CF Mouse Model

Background: High efficacy modulator therapy (HEMT) has revolutionized CF patient care significantly reducing patient lung function decline and quality of life, but not all CF patients can benefit from HEMT. An alternative approach, potentially curative, gene therapy, can treat CF regardless of patient mutation. Non-viral gene delivery is appealing as vectors can be designed to be safe and non-immunological. However, efficient delivery of nucleic acids with non-viral vectors in vivo has been difficult due to poor cellular uptake, extra- and intracellular barriers, and degradation. DNA nanoparticles (DNPs) are non-viral vectors shown to be safe in transfecting non-dividing cells in animals[1] and CF patients[2]. Previously, DNP interaction with nucleolin (NCL) at the cell surface was identified as an important mechanism for DNP cellular uptake and trafficking, and activating a NCL interactor, glucocorticoid receptor, enhanced gene transfer ~3 fold [3]. Based on data we hypothesized that manipulation of DNP interactors can significantly enhance gene delivery.

Methods: We transfected HeLa and primary airway epithelial cells with DNPs produced by compacting plasmid DNA with a 10kDa PEG-poly-l-lysine (PEG-Ck₃₀) conjugate. LC/MS of DNP immunoprecipitations identified 463 proteins, 79 interacting with NCL. We created a compound library that we predicted would modulate gene transfer. Three compounds, RX001, RX008, and RX011, were screened by reporter-gene analysis in HeLa cells and mice, and then tested in WT or S489X- mice to assess hCFTR transgene delivery to the lung. Mice were given an intraperitoneal injection of saline or compound 2 hours prior to DNP instillation (IT) into the lung. Mice that received luciferase DNPs had activity measured using BLI at 2, 3, 7 and 14 days after DNP administration. Mice that received hCFTR DNPs had lungs harvested 4 days after DNP administration for rtPCR, LC/MS, or western blot (WB) analysis.

Results: Two days after WT mice were given IT of luciferase DNPs following compound treatment, total photon output was 4.12, 6.09, and 5.88 fold higher in mice treated with RX001, RX008, and RX011, respectively, compared to DNP alone. Total photons were at least 2 fold higher at 3, 7, and 14 days in treated mice versus DNP alone. Four days after WT or S489X- mice received ITs of hCFTR DNPs, mRNA was isolated for rtPCR. ΔCt values for hCFTR were 5.30±0.24, 1.65±0.46, and 1.81±0.45 in mice that received DNP alone, RX001 or RX008, respectively. S489X- mice had ΔCt values of 11.68±0.37, 7.91±0.22, 3.01±1.23, and 4.93±0.98 in DNP alone, RX001, RX008, and RX011, respectively. To determine if hCFTR protein is present, S489X- lung lysates were prepped for LC/MS or WB analysis. 1.50±1.08 hCFTR peptides were identified in 5/8 DNP mice and 2.40±1.26 peptides were identified in 7/8 RX008 treated mice. No peptides were identified in the saline group. hCFTR was observed by WB in 5/8, 7/8, 0/8 mice treated with DNP alone, RX008, and saline, respectively.

Conclusions: Investigation of the DNP interactome can identify proteins that influence gene transfer and that modulation of proteins in the DNP interactome by small molecules can significantly enhance hCFTR gene transfer in a CF mouse model. Our data suggest that gene transfer modulators can enhance gene transfer in CF patients.

Acknowledgements: NIH R21EB023800
CFF RHEINE18F0

References: 1.Ziady et al; Mol. Ther. 2003;8(6):948-9562.Konstan et al; Hum. Gen. Ther. 2004;15(12):1255-12693.X. Chen, S. Shank, P. B. Davis, A. G. Ziady, Mol. Ther. 19, 93 (2011).