130. Purification of Large Scale mRNA Encoding ZFN Nucleases by dHPLC Technology

Abstract

A novel strategy of targeted gene correction of the interleukin-2 receptor common gamma chain (IL2RG) gene for the treatment of X-linked Severe Combined Immunodeficiency (SCID-X1) is achieved by the combination of a pair of IL2RG-specific Zinc Finger Nucleases (ZFN) and the correct-gene template DNA delivered by integration-defective lentiviral vector (IDLV).The transient expression of the ZFN pair targeting the disease-causing gene is obtained by the electroporation of the two corresponding mRNAs, produced by in vitro transcription starting from plasmid DNA template. A major limitation of the mRNA transcribed in vitro is the presence of residual contaminants such as short RNAs and double stranded (ds)RNAs that may affect the function and spectrophotometric quantification of the product hampering therefore the delivery of high quality and precise amount of mRNA to target cells. Moreover, dsRNA contaminants represent a possible risk in terms of immunogenicity of the product, leading to activation of unwanted innate immune response with consequent reduction/abrogation of mRNA translation as well as potential alteration of the properties of the transfected cells. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we combined different strategies: (i) inclusion of UTRs and polyA tails in the DNA template used for mRNA production; (ii) use of modified nucleotides during mRNA production and (iii) purification of the mRNAs by dHPLC with a reverse phase column made of non-porous matrix consisting of polystyrene-divinylbenzene copolymer beads alkylated with C-18 chains (Transgenomic, LTD.). In particular, the purification of in vitro transcribed mRNAs by means of dHPLC has been shown to strongly improve the translation of mRNA and significantly reduce the contaminant presence thus preventing innate immunity and eventually increasing modified cells persistence in vivo. We have developed feasible and reproducible, small and large scale mRNA production and downstream purification processes of the ZFN pairs obtaining accurate RNA quantification and reduced risk of immunogenicity. The full process achieved a 60% yield, loading with a 500µg RNA for each run with a single clean chromatographic peak. Furthermore, the level of residual organic solvent (i.e. Acetonitrile) used in the purification process is compatible with that applicable into clinic. The highly translatable non-immunogenic dHPLC-purified mRNA can be delivered without toxicity and represents a powerful and safe tool for the application of gene therapy protocols.