Figure 1

The CYP3A5*3 SNP Regulates Gene Splicing via a Salt-sensitive Mechanism that can be Modulated with ASO in Renal Cell Culture Models. (a) Schematic for the CYP3A5*3/*3 polymorphism found in HEK293 cells. The CYP3A5*3-SNP creates a cryptic splice acceptor within intron 3 that results in the aberrant splicing of a pseudo-exon containing a PTC that targets the mRNA transcript for degradation by NMD. Both the cryptic splice acceptor (CAG/TA) and the exon 4 splice acceptor (CAG/AA) are preceded by canonical U2 snRNP branch point cis-acting elements. (b) Endpoint PCR results monitoring exon 4 inclusion in mature CYP3A5 mRNA transcripts demonstrate the role of the *3-SNP in altering splicing and transcript stability. Wild-type CYP3A5 mRNA transcripts encoding exon 4 are virtually undetectable (no band) in HEK293 cell lysates under normal conditions, however when the *3-SNP site is masked by interactions with an antisense oligomer (CYP3A5*3 PMO; 3 µM; 48 h) transcript stability is restored, leading to enhanced detection of exon 4 inclusion via endpoint PCR. (c) Quantitative PCR (qRT-PCR; SYBR green) results demonstrating a 1.5-fold increase in total CYP3A5 mRNA when HEK293 cells were treated with the CYP3A5*3 PMO (1 µM; 48 h). (d) Quantification of the formation of 1-OH-midazolam from midazolam by LC/MS/MS after treatment with either 3A5*3 PMO or AUG PMO for 6 days in primary human PTECs. CYP3A5 activity was increased in PTECs treated with 3A5*3 PMO but not AUG PMO (**p < 0.01; t-test). Efficiency of restoration of CYP3A5 activity by the 3A5*3 PMO was donor-dependent (Donor 3; See Supplemental Table 1), and was not related to cellular toxicity, as PMO treatments did not alter normal PTEC morphology (see Supplemental Fig. 9).