Modified messenger RNA as a tool for ex vivo and in vivo gene correction
Final Report Abstract
Zinc-finger or transcription activator-like effector nucleases (ZFNs/TALENs) facilitate homologous recombination and represent a promising approach for repairing genomic mutations. Recently, an in vivo model of haemophilia was used to demonstrate nucleaseguided genome editing by using AAV vectors to encode for both the ZFN pair initiating the double strand break (DSB), as well as the donor template that facilitate homology-directed repair (HDR). While the use of stably expressing AAV vectors is documented to be safe, transient expression of the nuclease is sufficient for stable modification of the genome, making advanced, short-term ZFN expression strategies an advantageous approach. In recent years, modified messenger RNA (mRNA) has been pioneered, owing the potential as an alternative to traditional viral gene therapy vectors. While modified mRNA has been shown to provide a short half-life pulse of protein expression, it avoids also genomic integration. In addition, modification schemes can customize the stability of expression from these vectors and avoid immune activation even in mammals. Therefore, coupling nonviral delivery of nucleases with transient expression of mRNAs in vivo qualifies mRNA as an ideal delivery vehicle for genome editing nucleases. Here we utilize such optimized mRNA to deliver site-specific nuclease pairs to the lungs, to facilitate targeted gene correction in a mouse model of SP-B deficiency. To prove gene correction in the lungs, a panel of in silico designed ZFNs and TALENs was customized. Proxy dual-luciferase single-strand assay and T7-Endonuclease I-Assay confirmed TALEN-1 (T1) and ZFN-3 (Z3) to exhibit substantial cleavage efficiency, reaching up to 39 % of alleles repaired by non-homologous end-joining (NHEJ) ex vivo. Further analysis revealed also a significant increase in DSB-induction when encoded by mRNA versus Plasmid-DNA (pDNA, P < 0.05). In combination with a repair template designed to insert a novel NheI restriction enzyme site, T1 and Z3 also showed a significant increase in the efficiency of HDR compared to pDNA-encoded nucleases (P < 0.05). As Z3 was more efficient than T1 in both DSB-induction and HDR, Z3 nuclease was used for further experimentation. To optimize Z3 expression in an in vivo setting, two mRNA modification shemes (Ψ(1.0)/m5C(1.0) and s2U(0.25)/m5C(0.25)) were tested. Lung expression analysis following intratracheal (i.t.) administration of tagged Z3 mRNA, with or without complexation with Chitosan-coated poly(D,L-lactide-co-glycolide) nanoparticles (NPs), showed that s2U(0.25)/m5C(0.25) Z3 mRNA (referred to as Z3 nec-mRNA) complexed to NPs yielded the highest expression levels in both total lung cells and AT-II cells. Immunogenicity was monitored by IFN-α ELISA revealing no significant stimulation. Next, site-specific HDR using an AAV-encoded donor template (AAV6-donor), containing a constitutive CAG promoter, was efficiently transduced either with Z3-encoding AAV (Z3 AAV) or with Z3 nec-mRNA in primary fibroblasts. Therapeutic, genomic manipulation was also achieved in transgenic SP- B mice after delivery of donor and Z3 AAV or Z3 nec-mRNA, respectively, resulting in a significant prolonged survival compared to mock treated groups after doxycyclin removal (P < 0.001). Normal lung function was maintained in gene corrected mice with SP-B levels similar to that of positive controls with no apparent pathology. Compared to PBS-treated mice, ELISA for IL-12 expression revealed also no significant increase. Site-specific cleavage and HDR confirmed these phenotyic improvements, reaching similar HDR-levels in both Z3 AAV and Z3 nec-mRNA treated groups. Immunohistochemistry confirmed no expression of Z3 nec-mRNA after 20 days of treatment, whereas Z3 AAV was still detectable. The results confirm that gene targeting using nec-mRNA combined with an AAV-donor results in successful in vivo genetic engeneering of the transgenic SP-B locus, allowing constitutive expression of SP-B, improved lung function, and prolonged survival in the abscence of doxycyclin. Compared to AAV, nec-mRNA provided a shorter burst of nuclease expression, which may lessen the threat of ongoing cleavage activity. Furthermore, due to their ability to be non-immunogenic, repeated administration of modified mRNA to the lung can be applicable. In conclusion, nec-mRNA is an important step in the development of clinically relevant, therapeutic gene correction strategies for the treatment of inherited lung disease and other monogenetic diseases. http://www.bio-pro.de/magazin/index.html?lang=en&artikelid=/artikel/10062/index.html http://www.labhoo.com/PressImage/2014/PR_8054.htm http://www.lungenaerzte-im-netz.de/lin/linaktuell/show.php3?id=2731&nodeid=18 http://news.doccheck.com/de/88467/atemnotsyndrom-per-nanofaehre-durch-die-galaxis/
Publications
- (2013). "Modified mRNA encoding Foxp3 protects against allergic asthma in mice through an IL-23/IL-17A-dependent mechanism." J Clin Invest 123(3): 1216-28
Mays LE, Ammo-Treiber S, Mothes B, Müller-Hermelink E, Grimm M, Mezger M, Beer- Hammer S, Nürnberg S, Schwab M, Handgretinger R, Idzko M, Hartl D and Kormann MSD
- Flagellin incudes myeloid-derived suppressor cells: Implications for Pseudomonas aeruginosa infection in Cystic Fibrosis Lung Disease. The Journal of Immunology, Vol. 190, Issue 3, 1 Feb 2013, 1276-1284
Rieber N, Brand A, Hector A, Graepler-Mainka U, Ost M, Schäfer I, Wecker I, Wirth A, Neri D, Mays LE, Zundel F, Handgretinger,Fuchs J, Stern M, Hogardt M, Döring G, Riethmüller J, Kormann MSD, Hartl D
(See online at https://doi.org/10.4049/jimmunol.1202144) - "In vivo gene correction of Cystic Fibrosis" as part of the book: "Cystic Fibrosis in the Ligth of New Research" (2015, ISBN 978-953-51-4275-1)
Kormann MSD
- (2015). "In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency." Nat Biotechnol Epub 2015 May 18
Dewerth A, Mahiny AJ, Mays LE, Alkhaled M, Mothes B, Malaeksefat E, Loretz B, Rottenberger J, Brosch DM, Reautschnig P, Surapolchai P, Zeyer F, Schams A, Carevic M, Bakele M, Griese M, Schwab M, Nürnberg B, Beer-Hammer S, Handgretinger R, Hartl D, Lehr CM and Kormann MSD
(See online at https://doi.org/10.1038/nbt.3241)
