PhiC31 Integrase as a Candidate for Retinal Gene Therapy

Abstract

Abstract: : Purpose: The integrase protein from the phiC31 phage directs site-specific recombination between two sequences: an attP site on the phiC31 phage and an attB site on the Streptomyces genome. This reaction does not require cofactors, and can proceed efficiently in vitro. Mammalian genomes contain endogenous sites with partial identity to attP (termed pseudo attP sites). PhiC31 integrase can mediate recombination between such sites and attB (1). We aim to use the phiC31 integrase as a tool for ocular gene therapy and will first advance the technology in animal models of retinal degeneration. Methods: In order to establish whether phiC31 integrase could be effective for in vivo gene therapy, the human FIX transgene (with or without the integrase gene) was delivered to mouse liver cells by using a high-pressure tail vein injection of plasmid DNA. For retinal delivery, we inject a DNA-liposome complex subretinally. Results: In mouse liver, co-injection of integrase led to 10-fold higher hFIX serum levels that remained stable for over eight months. Site-specific integration was proven by characterizing and quantifying genomic integration at the DNA level (2). Having established in vivo gene transfer by site-specific genomic integration, we seek to advance the integrase technology in the mammalian retina. We are currently investigating the potential of integrase-mediated transgene delivery in several models of retinal degeneration. Conclusions: These properties make the phiC31 integrase a powerful tool for chromosome engineering. By placing a therapeutic gene on a plasmid bearing attB, permanent site-specific integration into the mammalian genome can be achieved. Because site-specific integration affords strong, stable expression while reducing the probability of harmful insertion, the integrase system represents a novel approach to non-viral gene therapy with great potential. References:1. Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP. "Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase." Mol Cell Biol., 2001; 21: 3926-3934. 2. Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP. "Site-specific genomic integration produces therapeutic Factor IX levels in mice." Nature Biotechnology, 2002; 20: 1124-112.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary