195. Comparative Analysis In Vitro of Regulatory Elements That Drive Targeted Gene Expression in Adenovirus-Associated Viral (AAV) Vectors

Abstract

Adeno-associated viral (AAV) vectors are a platform with great potential for therapeutic gene delivery. For neurodegenerative disorders such as Friedreich's ataxia, identifying a gene therapy candidate that provides clinically relevant expression of the therapeutic transgene is critical. Friedreich's ataxia is caused by the decreased expression of the mitochondria matrix protein frataxin (FXN) due to mutations in the frataxin gene. Elevation of FXN protein in the nervous system is an approach that is likely to be beneficial in addressing the neurological components of this devastating disease. However, the properties of regulatory elements that drive exogenous gene expression from AAV vectors have not been well characterized. In this study, a set of AAV vectors were generated with various combinations of different regulatory elements directing the expression of the human frataxin gene. These regulatory elements included the canonical Kozak sequence, mitochondria targeting sequences, simian virus 40 (SV40) intron, AAV inverted internal repeat (ITR), and constitutive promoters such as cytomegalovirus (CMV), chicken b-actin (CBA), human frataxin, human phosphoglycerate kinase-1 (PGK), human synapsin and human elongation factor 1α (EF-1α) promoters. For comparison in vitro, these AAV constructs were introduced into three types of human cells and primary rat DRG neurons by co-transfection with a GFP expression construct which was used as an internal control. FXN and GFP protein expression were quantitatively measured by ELISA and the relative level of FXN expression calculated by normalizing to the level of GFP in each condition. These studies demonstrated that the CMV promoter is the most potent in driving FXN expression followed by the CBA promoter. Much lower FXN expression levels were observed when the endogenous frataxin or PGK promoter was used. The canonical Kozak sequence, the SV40 intron alone and different mitochondria targeting sequences had no significant impact on FXN expression. In addition, we evaluated the effect of different optimized human codons within the frataxin transgene on FXN expression level in the context of the CBA promoter and SV40 intron. Our results demonstrated that several of these codon-optimized frataxin genes consistently increased the expression of FXN protein at least 2-fold compared to the wild type frataxin gene. Together, these results provide important information for guiding the selection and optimization of regulatory elements and codons for an AAV gene therapy for Friedreich's ataxia.