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BREATHE Report Summary

Project ID: 637752
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - BREATHE (Biochemically modified messenger RNA encoding nucleases for in vivo gene correction of severe inherited lung diseases)

Reporting period: 2015-04-01 to 2016-09-30

Summary of the context and overall objectives of the project

Following the development of novel modified messenger RNA (mRNA)-based gene therapy and gene correction treatment approaches in a mouse model of the severe inherited lung disease, SP-B deficiency, I would like to expand these findings in the immediate future to a humanized mouse model of SP-B deficiency that is directly translatable to clinical trials. By creating a humanized mouse model of this rare genetic disease, we can optimize therapeutic approaches capable of correcting the animals’ disease phenotype in a setting that allows efficient translation of the therapeutic agents from bench to bedside.

From there, I’d also like to expand my laboratory’s findings further to the development and correction of humanized models of the more common lung disease, Cystic Fibrosis (CF), also with the aim of clinical translation. In our proposed, humanized CF model, we will administer a therapeutic approach to correct several prominent genetic defects that are commonly responsible for CF disease phenotype in patients. Several phenotypic outcomes will be followed as a measure of disease correction. For instance, as patients with CF lung disease are more susceptible to bacterial infection in the airways, we will infect mice intratracheally with P. aeruginosa and Burkholderia cepacia (B. cepacia) complex and monitor bacterial load over time. With this challenge, only mice whose genetic defect has been corrected will be able to survive the infection.

The overall objectives are:

1. Optimization of nuclease-encoding chemically modified mRNA (nec-mRNA)-based gene correction strategies that are directly translatable to clinical trials
2. Development, characterization, and treatment of a humanized mouse model of SP-B deficiency, including: the investigation of SP-B phenotype, in vivo gene correction of disease, and confirmation of disease correction
3. Development, characterization, and treatment of a humanized mouse model of cystic fibrosis containing the class II mutation, F508, including: the investigation of bacterial susceptibility and CF immune phenotypes, in vivo gene correction of disease, confirmation of disease correction, and analysis of bacterial killing

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Following key experimental designs AND respective achievements have been reached so far:

1. Using different modifications, tested in different cell lines, we could evaluate the best modifications to either peak the expression to high levels within a short period of time (24-48h), or to increase the expression pattern for more than 5 days.
Thus, having different modifications available, we can now control the time a specific gene (in this case a site-specific endonuclease such as TAL Effector Nuclease or CRISPR/Cas9 systems) will be expressed. This leads to the next goal for which we do in vivo tests with modified Cas9 mRNA to 1) evaluate the most efficient modification and to 2) reach efficient cutting efficiency for HDR at the SP-B STOP mutation site and the CFTR delF508 mutation site

2. We successfully could create functional sgRNA that binds to and cuts at the target sites; in addition, we created ssODNs as a template to either insert a restriction tag as a prove of principle or to insert the correct version at the mutation site.
Although cutting efficiency is still relatively low, recent advances in modifying the Cas9 mRNA as well as ssODNs might enhance the NHEJ as well as HDR. We are about to test that in the upcoming months in vitro and in vivo, together with in vitro encapsulating the mRNA in different Nanoparticle formulations.

3. To detect expression of reporter and SP-B / CFTR in lung stem cells, and gene correction events, we are in the process of setting up an antibody panel to directly have a prove for mRNA Cas9 /expression in lung stem cells. With the available equipment, we are able to define - besides lung epithelial cells - also lung stem cells at the same time using an antibody panel with up to 18 colors which gives us a great opportunity to assess the effect with respect to different targeting strategies

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

"1. By comprehensively screening the different kinetic profiles of mRNA encoded endonucleases, we have a significantly higher efficacy protocol for in vivo gene editing as well as a significantly lower chance for off-target effects.
2. The combination with certain nanoparticles (in cooperation with HelmholtzZentrum Saarbrücken) we do now not only have the intratracheal spray application as primary application method, but also the i.v. route - which is a huge advantage to reach lung (stem) cells - as natural ""defense"" systems like cilia or disease-correlated symptomatic issues such as mucus can be circumvented.
3. In comprehensive analyses we are understanding mRNA immunogenicity and how to avoid or completely abrogate that. This will be mandatory for first in men studies.

(Evtl aus dem Grant noch Formulierungen verwenden...)"
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