Novel Modalities, Novel Delivery
Properly skilled and trained research professionals are paramount to stimulate ongoing innovations in viral vector-based gene therapy development, and to ensure their effective implementation in industrial and clinical settings. As explained below, each Doctoral Candidate (DC) has a specific assignment with a dedicated focus.
created with BioRender.com
In WP2, GET-IN will develop several innovative modalities for gene therapy to address these challenges. We will develop novel programmable gene editing nucleases for human applications, with the aim to at least equal the efficiency of CRISPR-Cas9 but without the identified safety issues. In addition, CRISPR base and prime editing will be engineered and tested for the first time in in vitro human lung models. These novel gene editing approaches will be combined with novel delivery systems (Virus Like Particles (VLPs), VesiCas) allowing transient delivery ensuring tissue-targeted nuclease conveyance. For delivery to specific tissues or cell types, and to uplift gene transfer technology from ex vivo to in vivo, the membrane of the VLPs will be functionalized employing pseudotyping by envelope glycoproteins.
A large variety of yet uncharacterized CRISPR-Cas nucleases still reside unexplored in the human gut microbiome. DC6 will interrogate a metagenomic assembly of unexplored programmable nucleases from the gut microbiome. Novel nucleases will be tested for activity, and best candidates further characterized for editing efficiency, off target activity and compatibility with delivery systems developed within GET-IN.
VesiCas is based on extracellular vesicles for the traceless delivery of protein cargo, such as genome editing tools. This technology will be extended towards neuronal cells/central nervous system (CNS) as a new cell- or tissue-targeted delivery vehicle for genome editing. DC9 will identify novel CNS targeting pseudotypes, develop a cell-based screening system to allow streamlined identification of candidates with the desired tropism for targeted delivery. Cargo flexibility for different genome editors (base editors, prime editors, epigenome editors) as well as novel CRISPR-Cas nucleases will be investigated (including editors developed by DC2, DC6) and benchmarked.
Nanoblade (NB) technology for delivery of the CRISPR-Cas9 system into primary HSC will be explored. NB are genetically modified retrovirus-derived virus like particles loaded with Cas9 protein and guide RNAs. DC5 will transfer the technology developed for retargeting LVs to the NBs, using cell specific binding moieties (e.g. antibodies, DARPins, envelope glycoproteins) to target T cells, macrophages and HSC. Testing will be done in patient derived in vitro models, in the OoC models of WP3, and humanized mouse models available at INSERM.
Gene therapy is being investigated as a possible treatment for the 15% of cystic fibrosis (CF) patients that do not respond to therapy. Effective delivery to airway epithelial cells and rapid loss of transgene expression are major obstacles. We demonstrated correction of specific CFTR (CF transmembrane conductance regulator) mutations by CRISPR-Cas gene editing in cell lines and patient-derived rectal organoids. As a next step, DC2 will explore translational delivery of gene editing machinery and evaluation of efficacy and safety in relevant in vitro human lung models. The DC will develop safe and efficient delivery of prime editing machinery by NB and VesiCas (in collaboration with DC9).