In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency

Abstract

In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency (2015) Nature Biotechnology, 33 (6), pp. 584-586.In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiencyTo the Editor:Nuclease-mediated genome editing holds great potential to knock out or repair diseasecausing genes. An ideal nuclease delivery vehicle is short-lived, does not integrate into the genome, and can enter target cells efficiently. These requirements have not yet been achieved simultaneously by any nuclease delivery vector. We and others have used modified mRNA, which is non-integrating and provides a transient pulse of protein expression, as an alternative to traditional viral vectors [1][2][3][4][5] . This approach allowed us to deliver therapeutic proteins in mouse models of Surfactant Protein B (SP-B) deficiency 3 and experimental asthma 4 . Here we apply it to deliver site-specific nucleases, demonstrating the value of nuclease-encoding chemically modified (nec) mRNA as a tool for in vivo genome editing. We chose a well-established transgenic mouse model of SP-B deficiency 6 in which SP-B cDNA is under the control of a tetracycline-inducible promoter 7 . Administration of doxycycline drives SP-B expression levels similar to those in wild-type mice (Supplementary Fig. 1), whereas cessation of doxycycline leads to phenotypic changes similar to those of the human disease, including thickened alveolar walls, heavy cellular infiltration, increased macrophages and neutrophils, interstitial edema, augmented cytokines in the lavage, a decline in lung function, and fatal respiratory distress leading to death within days 8,9 . We inserted a constitutive CAG promoter immediately upstream of the SP-B cDNA to allow doxycycline-independent expression and prolonged life in treated mice.First, we customized a panel of ZFNs and TALENs targeting the transgenic SP-B cassette ( Fig. 1a and Supplementary Fig. 2). We chose TALEN #1 (T1) and ZFN #3 (Z3) owing to their high activity and proximity to the desired site of promoter integration (Figs. 1a,b; amino acid sequences in Supplementary Fig. 4) and compared delivery by plasmid 1 DNA and mRNA. mRNA delivery resulted in higher levels of double-strand break (DSB)-induction ( Fig. 1c and Supplementary Fig. 3; P < 0.05) and homology-directed repair (HDR) ( Fig. 1d, P < 0.05). As Z3 mRNA was more efficient than T1 mRNA in both cases, Z3 was chosen for further experimentation. Comparison with a Z3-encoding AAV serotype 6 vector (AAV6) ("Z3 AAV") shows the relatively transient expression of Z3 mRNA (Fig. 1e), limiting the time during which off-target cleavage activity could occur.To optimize Z3 expression in the mouse lung, we administered a panel of 3xFLAG-tagged Z3 mRNAs with various modification schemes 2,5,10 , with or without complexation to biocompatible, biodegradable nanoparticles (NPs) made of chitosan-coated poly (lactic-coglycolic) acid (chit-PLGA) 11,12 . Following intratracheal (i.t.) delivery, NP-complexation significantly increased mRNA expression levels ( Supplem...

Featured Article: Electroporation-mediated gene delivery of surfactant protein B (SP-B) restores expression and improves survival in mouse model of SP-B deficiency

“…A second study from the same group developed a novel zinc finger nuclease to target the SP-B cassette and delivered this to the lung as a modified mRNA using chitosan-coated poly(lactic-co-glycolic) acid nanoparticles. 31 When delivered intratracheally along with Adeno-Associated virus 6 carrying a wild type copy of the intact SP-B gene as a donor template, gene correction was achieved and resulted in high SP-B protein levels for at least 20 days and enhanced survival for out to 35 days. While this approach did not elicit significant inflammation, the combined use of multiple delivery methods (non-viral and viral) could be cumbersome or a regulatory difficulty.…”

“…In one, modified mRNA for hSP-B was used as a treatment for SP-B deficiency in the SP-B compound knockout mouse model used in the current study and had very promising results. 30 Kormann and his co-workers 31 delivered modified mRNA for hSP-B to the lungs of the SP-B compound knockout mice via an intratracheal spray method. By performing gene delivery twice weekly, survival improved up to 28 days off doxycycline.…”

Impact of gene editing on the study of cystic fibrosis

“…Lentivirus vectors have even been used to deliver the ZFN or TALEN proteins and edit cells (Cai et al 2014), and more recently, direct delivery of Cas9/ gRNA ribonucleoprotein complexes has been described (Choi et al 2016). Of particular interest for CF, the combination of non-viral and viral delivery, specifically the use of chemically modified mRNA encoding site-specific nucleases delivered with chitosan nanoparticles, and AAV to deliver the donor, resulted in precise editing in the lungs, within a notable phenotypic change in a well-established transgenic mouse model of surfactant protein B deficiency (Mahiny et al 2015). The availability of large animal CF models has also established the feasibility of delivery of aerosolised virus vectors intratracheally into pigs under bronchoscopic guidance (Cao et al 2013;Yan et al 2015).…”

Gene Editing and Genetic Lung Disease. Basic Research Meets Therapeutic Application

“…Furthermore, progeny of the genetically corrected mice were free of cataracts. Thus, multiple studies have (38). However, the limitation of this model is that it is a compound transgenic mouse model in which SFTPB is expressed conditionally and does not ideally represent the disease-causing mutations found in humans (39).…”