Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy

Abstract

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a qRT-PCR method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, total HO-1 mRNA was enriched 2-fold relative to control cells and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery and monitoring of gene therapy vectors.

Heme oxygenase-1 gene delivery by Sleeping Beauty inhibits vascular stasis in a murine model of sickle cell disease

“…For in vivo experiments in mice, a rat hmox-1 pBluescript plasmid containing an SV40 enhancer, a Friend's Spleen Focus-Forming Virus LTR promoter, the entire coding region of the wild-type (wt) rat hmox-1 gene, and SV40 polyadenylation [16] was blunt cloned between two direct repeat (DR) binding sites within two inverted repeat (IR) sites (IR/DR) into a 2-kb, albumin (Alb)-driven, SB 10 transposase plasmid [12]. The IR/DR elements act as essential binding sites for the transposase [17].…”

“…Clones were screened by restriction digest mapping and sequencing followed by protein expression in tissue culture, HO-1 western blot analysis, and HO enzyme activity. The resultant clone ( SB -wt-HO-1, 5,793 bp) expressed wt rat HO-1 (wt-HO-1) as previously described [12]. …”

“…Total RNA was isolated from frozen organ sections, and HO-1 mRNA was quantitated by quantitative real-time polymerase chain reaction (qRT-PCR) as previously described [12]. The presence or absence of the wt- or ns-rat HO-1 transgene was confirmed by restriction fragment length polymorphism (RFLP) analysis of HO-1 cDNA using ApaI restriction enzyme digestion as previously described [12].…”

“…To transfer the rat HO-1 gene into mice, we cloned the rat hmox-1 gene into a cis Sleeping Beauty transposase ( SB -Tn) system with the transposase driven by an albumin promoter [12]. The SB -Tn system was developed by resurrecting nonfunctional remnants of an ancient vertebrate transposable element from salmonid fish [13].…”

Heme Degradation and Vascular Injury

“…Increases in HO-1 activity or its downstream products or both, in excess of the adaptive upregulation seen in tissues of sickle cell mice and patients, may be important strategies for innovative new therapies to prevent and treat hemolysis, oxidative stress, inflammation, vasoocclusion, and the accompanying pathologies found in sickle cell disease. HO-1 gene therapy, by using Sleeping Beautymediated transposition of an HO-1 transgene, is a promising nonviral approach to enhance HO-1 expression significantly in sickle cell disease (34). To transfer the rat HO-1 gene into sickle mice, our laboratory has cloned the rat hmox-1 gene into a Sleeping Beauty transposase (SB-Tn) system.…”