Introduction

For most monogenic diseases, treatments are either not available or merely symptomatic. Furthermore, the widespread use of CRISPR-Cas9 based therapies is hindered by the risk of non-specific genome alterations (off-target activity). The proposed CRISPR-Cas9 system characterized by a reduced off-target activity consists of two viral particles encoding a guide RNA (gRNA) recognizing the region of the mutated DNA, and a donor sequence as template to correct the mutation. The above system is applicable in personalized gene replacement therapy of monogenic diseases such as Pompe disease, Rett syndrome, Alport syndrome and Parkinson disease.

Technical features

The therapeutic CRISPR-Cas system is encoded in two separate vectors, one for the Cas9 endonuclease and the other for the donor DNA, each encapsulated in distinctive viral particles. The engineered viral particles vehiculate both vectors into non-dividing target cells, such as kidney and neurons. Once transduced, the DNA coding for the cas9 and gRNA are transcribed and, for the former only, translated. Through the gRNA, Cas9 recognizes the mutation responsible for monogenic disease and cleaves the mutated locus in the host genome. Subsequently, the donor DNA in the second vector operates as a template for site-directed recombination and “corrects” the mutation. Finally, the gRNA-Cas9 complex self-inactivates by digesting its encoding vector through the same recognition and cleavage mechanism as above, for the vector also contains a recognition site for gRNA. This hit-and-run mechanism thus avoids the problem of off-target genomic cleavage. The technology, (TRL= 4), can be used for the treatment of rare genetic diseases characterized by somatic mutations in non-replicating cells, such as kidneys (Alport syndrome) or neurons (Rett syndrome, Parkinson’s disease).

Possible Applications

• Personalized medicine, and in particular for the treatment of monogenic diseases such as Alport’s syndrome, Pompe’s disease, Rett’s syndrome and Parkinson’s disease.

Advantages

• Automatic inactivation of the system through recognition sequences for gRNA directly on the vector coding the Cas9 endonuclease;
• Risk reduction for non-specific genome restriction;
• System designed for targeting non-replicating cells (e.g. neurons or kidneys), allowing gene replacement therapy in otherwise hard-to-reach target cells;
• System components used in different routes of administration and applied to other diseases subject to limited modifications.