270. Nanoparticle-Mediated Rhodopsin cDNA But Not the Intron-Containing DNA Delivery Causes Transgene Silencing in a Rhodopsin Knockout Model

Abstract

Gene expression is moderated by a multitude of patterns. The cross-talk between the native promoters and all the non-coding endogenous elements, such as transcription factors, enhancers and introns, are tightly controlled in a precise and real-time manner.The use of cDNA transgene often causes transgene silencing as over-expression or under-expression is known to affect protein function. In contrast, large genomic DNA transgene showed positionindependent, copy number-dependent expression of the transgene. Precise control of rhodopsin expression is critical in maintaining viable photoreceptors. In the present study, two forms of rhodopsin DNAs, an intron-containing short form genomic DNA and an intron-less cDNA, were cloned into an S/MAR vector driven by the 500 bp mouse opsin (MOP) promoter. The vectors were further compacted into nanoparticles (NPs) and subretinally injected into a rhodopsin knockout mouse (RKO) model at postnatal day 3. Following injection, DNA methylation and histone status of the specific regions (bacteria plasmid backbone, promoter, rhodopsin gene and S/MAR) in the vectors were evaluated at various time points. Persistent expression and phenotypic improvement were found in RKO mice up to 8 months after NP-mediated intron-containing delivery but not the NPs housing the intron-less cDNA delivery (data submitted for publication under review). In this study, our results further indicated that epigenetic silencing occurred in vector-mediated gene transfer, which were contributed by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene as evaluated by DNA methylation specific PCR followed by DNA sequencing and chromatin immunoprocipitation (ChIP) assay with highly specific antibodies for euchromatin and heterochromatin. No toxicity or inflammation was found in the treated eyes as detected by both PCR for pro-inflammatory cytokines and immunohistochemistry using macrophage marker, F4/80. These results suggest that cDNA of transgene and bacteria backbone interfere with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochratin-associated modification. While the benefits of including genomic elements may vary from gene to gene, our data suggest reconsider the current paradigm using cDNA as the standard in gene delivery cassettes. This paradigm shift has wide-ranging application for ocular gene therapy for retinal diseases or for which a genetic treatment is of interest. Further experiments are currently underway to determine whether inclusion of additional endogenous elements will provide added advantages in terms of physiologically relevant gene expression.