Differentiation of Highly Conserved Host and Transgene mRNA in HO-1 Gene Therapy.
Authors: Bruzzone Carol M. , Newman Kristal A. , Belcher John D. , Steer Clifford J. , Vercellotti Gregory M. 
Published: April 2020

Background: Heme oxygenase-1 (HO-1) is highly inducible in vitro and in vivo and appears to confer protection to cells and tissues from oxidative stress and inflammation. Marked oxidative stress and vascular inflammation are hallmarks of sickle cell disease (SCD). We have previously shown that enhanced expression of HO-1 inhibits vascular inflammation and hypoxia-induced vaso-occlusion in murine models of SCD. To further these observations, we constructed transgene forms of rat HO-1 for gene therapy in murine models of SCD. The high degree of interspecies sequence conservation makes epitope choices for primary antibody and nucleic acid sequence available for probes or primers problematic for differentiation of endogenous and transgenic HO-1. There are only 16 differences in the 289 amino acids between murine and rat HO-1. At the genetic level, murine and rat HO-1 are 93% identical. We utilized interspecies RNA sequence differences to accurately differentiate endogenous murine HO-1 from rat transgenic HO-1.

Methods: Two pairs of RT-PCR primers were designed from areas of interspecies identity. Both primer pairs generated cDNAs with 10 base pair differences between species. In addition, each flanked an interspecies single nucleotide difference, which represents a unique restriction site in the rat, but not the murine cDNA. The GGGCCC sequence in rat is an ApaI restriction endonuclease (RE) site whereas the murine GGGCCT is not. The second primer pair created an RE site through site specific primer induced restriction fragment length polymorphism (SSPI RFLP). Rat sequence at bp 375 is TCCTGA; murine is CCCTGA; and RE BspEI recognizes TCCGGA. Our primer incorporates a mismatch in its third position, modifying the PCR product to TCCGGA and CCCGGA, respectively. This rat cDNA can be digested with either ApaI or BspEI. PCR amplification of HO-1 was performed on quantified, DNAseI treated, ApaI digested total RNA with either primer pair in a 25ul one tube system that included RNase inhibitor.

Results: The quantification of total HO-1 RNA relative contribution from each species was performed by densitometry via Molecular Analyst™ Program on 2% agarose gel electrophoresis separation of restriction enzyme digested total PCR product. Murine PCR products were 200 bp as they do not contain either restriction site. ApaI and BspEI digestion of rat PCR products produced bands at 130 bp and 70 bp or 175 bp and 25 bp, respectively. Control experiments utilizing liver or spleen total RNA from both species demonstrated that mixed samples produce digested cDNA that reflects their relative proportion by species and that the reaction is reliable in dilution series.

Conclusions: RFLP and SSPI RFLP can be used to differentiate highly conserved transcripts of native HO-1 RNA from transgene HO-1 RNA. RFLP and SSPI-RFLP can be applied to discriminate native inducible transcripts from transgenes in gene therapy studies, when activity, Western blot and immunofluorescence are not viable options. We are using this novel methodology in our ongoing studies using rat transgenes in murine models of SCD.