Abstract. The choice of a delivery system for genetic manipulation or gene expression remains a topic of debate in the research community. The development of a safe, unique, and efficient delivery vehicle that could promote gene editing would significantly contribute to the field of gene therapy and regenerative medicine. Recently, we have demonstrated that the Measles virus can be developed as a single cycle vector for multiple gene delivery. We have shown that we can express the four reprogramming factors from on single measles genome and reprogram adult human fibroblasts into induced pluripotent stem cells. We further have evidence that MeV can express the gene-editing system CRISPR-Cas9 and lead to the correction of cells in the presence of a molecule of DNA template. The goal of this application is to develop new prototype measles vectors that can either genetically modify or correct a specific gene locus without relying on a DNA molecule using Prime Editors. As a proof of concept, we will develop prototype vectors based on the vaccine strain of the measles virus, a negative-strand RNA virus. The objectives of this application are (1) to establish the proof of principle that the MeV vector can express the Prime Editing system (MeV-PE2). To do this, we will express the two elements required for prime Editing: the Prime editor (PE2) and prime editor guide RNA (pegRNA), and test their functionality in producing modified cells for the correction of Sickle cell disease (SCD) (2) In the second aim, we will introduce the latest generation of Prime editor in MeV vector (MeV-PEmax), which consists of the PE2 and the introduction of (i) a standard gRNA targeting the non-edited template, (ii) the MLH1dn protein to reduce mismatch repair or (iii) a modified pegRNA to increase its stability. The proposed work is innovative, challenging, and significant, and if successful, it will lead to the production of novel prototypes of “all-in-one” non-integrating RNA viral vectors for cellular gene editing without the addition of an exogenous molecule of DNA. This work will open the future development of safe vectors for gene editing and the production of corrected cells for multiple genetic disorders. This new platform will have a significant impact in the field of gene therapy and regenerative medicine.

Narrative The proposed research will have a high impact in the field of medicine, as it will develop new prototype vectors able to deliver the Prime Editor system efficiently, leading to the production of safer modified cells. In addition, this research is relevant to public health, and it will have a meaningful impact on developing new regenerative medicine and gene therapy approaches to treat monogenic diseases and other genetic diseases.