• Du 01 janvier 2021 au 31 mai 2021
    22 Bd Benoni Goullin
    44200 Nantes
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  • 2-month internship will be held between January and May 2021

Plasmid design and cloning for the generation of new AAV viral vectors

Adeno-Associated Viral Virus (AAV) are one of the most suitable viral vectors for gene delivery in vivo. Encouraging preclinical data using AAV vectors have been followed by several successful gene therapy clinical trials and by the marketing authorization of two AAV-based products, Glybera® and Luxturna®. The scientific community is now facing news issues addressing systemic diseases such as Duchenne muscular dystrophy which requires a high dose of vectors. In addition to manufacturing limitations, injecting more than 1014 AAV particles per kg may trigger unwanted side effects such as an immune response to the viral capsids and the recombinant genome. Cell sensing of the exogenous DNA can reduce the expression of the therapeutic gene and impair the gene therapy treatment. Thus, it appears essential to optimize the recombinant AAV genome in order to reduce cell restriction and innate immune response to DNA.

The AAV genome is a single-stranded DNA of ≈4.7 kb composed of three open reading frames, rep, cap and AAP. To design a recombinant AAV vector, these ORFs are replaced by the therapeutic gene. The only viral sequences that remain in the vector genome are the ITR (inverted terminal repeats). ITR are 145 base-sequences comprising palindromic regions that can form a T-shaped structure (Figure). These sequences can be recognized by the cellular sensing pathways after transduction.


In our team, we have designed new ITR sequences in order to avoid this cellular response. The M1 student will participate to the design of the plasmids necessary to generate the new AAV vectors. The student will clone, amplify and verify these plasmids. He will eventually test the plasmids for the production of AAV viral vectors. The candidate must be interested in molecular biology, virology and cellular sensing pathways. He will take part of a more general project on the optimization of recombinant AAV genomes. Qualities such a rigor, organization and motivation will be appreciated.

Keywords : gene therapy, adeno-associated virus, viral vectors, inverted terminal repeats, molecular biology

Contact : Magalie Penaud Budloo