103. Pas de Deux – A Single Inducible High-Capacity Adenoviral Vector for Delivery of a Functional TALEN Pair Directed Against Human Dystrophin for the Treatment of Duchenne Muscular Dystrophy (DMD)

Abstract

Transcription activator like effector nucleases (TALENs) are powerful tools for gene therapeutic approaches that aim to induce site specific double-strand breaks (DSB) at the desired DNA locus. These DSB are usually repaired in the cells by non-homologous end joining leading to insertions or deletions (indels) causing mutations at the respective DNA locus. In the presence of homologous DNA sequence the DSB can be repaired by homologous recombination. It has been shown previously, that delivery of functional TALENs through viral vectors is feasible. Since TALEN genes are relatively large, previous viral delivery approaches for TALENs were based on two single viruses each carrying one TALEN gene of a respective TALEN pair requiring co-infection with two viruses. Here we report the production and functional analysis of a single gene deleted high-capacity adenoviral vectors (HCAdV) for the delivery of a complete TALEN pair (TN3/TN8) directed against the human dystrophin gene for the treatment of Duchenne muscular dystrophy. During virus production constitutive expression of a TALEN pair where the respective genes were controlled by CMV promotors viral amplification was strongly inhibited. Therefore we constructed a single HCAdV (HCAdV-Tre-TN3-Tre-TN8) carrying the TN8 and TN3 gene under the control of the inducible Tre promotor as well as two HCAdV each carrying a single TALEN gene under control of the CMV promotor (HCAdV-CMV-TN3 and HCAdV-CMV-TN8). We successfully delivered the TN3/TN8-TALEN pair to HEK293 cells and muscle cells either with co-infection of constitutively expressing single TALEN viruses or a single infection with the inducible double-TALEN virus. Independent of the delivery approach q-PCR based amplification refractory mutation detection showed substantial mutation rates at the genomic target locus of infected cells. At the same multiplicity of infection a single infection with the double TALEN HCAdV-Tre-TN3-Tre-TN8 caused similar mutation rates as co-infection with the single TALEN HCAdV-CMV-TN3 and HCAdV-CMV-TN8. The efficacy of the double-TALEN approach may be due to the delivery of a complete TALEN -pair using a single vector, whereas co-infection needs two vectors and cells may only receive a single TALEN gene being inactive without its counterpart. Infection with a single virus to deliver a complete TALEN pair is advantageous since the amount of virus could be reduced to achieve the same results than the co-infection approach. The usage of an inducible expression system may also provide a better safety profile for TALEN-based gene correction approaches by avoiding off target site cleavage caused by continuous nuclease expression. Therefore inducible HCAdV vectors for the delivery of functional TALEN pairs are promising tools for future TALEN- based gene therapy strategies.