Antisense oligonucleotide development for the selective modulation of CYP3A5 in renal disease

Kevin A Lidberg,Maria Beatriz Monteiro,Jonathan Himmelfarb,Andrew J Annalora,Susanne Ramm,Patrick L Iversen,Marija Jozic,Lu Wang,Theo K Bammler,Edward J Kelly,Daniel J Elson,Craig B Marcus

doi:10.1038/s41598-021-84194-w

Abstract

CYP3A5 is the primary CYP3A subfamily enzyme expressed in the human kidney and its aberrant expression may contribute to a broad spectrum of renal disorders. Pharmacogenetic studies have reported inconsistent linkages between CYP3A5 expression and hypertension, however, most investigators have considered CYP3A5*1 as active and CYP3A5*3 as an inactive allele. Observations of gender specific differences in CYP3A5*3/*3 protein expression suggest additional complexity in gene regulation that may underpin an environmentally responsive role for CYP3A5 in renal function. Reconciliation of the molecular mechanism driving conditional restoration of functional CYP3A5*3 expression from alternatively spliced transcripts, and validation of a morpholino-based approach for selectively suppressing renal CYP3A5 expression, is the focus of this work. Morpholinos targeting a cryptic splice acceptor created by the CYP3A5*3 mutation in intron 3 rescued functional CYP3A5 expression in vitro, and salt-sensitive cellular mechanisms regulating splicing and conditional expression of CYP3A5*3 transcripts are reported. The potential for a G-quadruplex (G4) in intron 3 to mediate restored splicing to exon 4 in CYP3A5*3 transcripts was also investigated. Finally, a proximal tubule microphysiological system (PT-MPS) was used to evaluate the safety profile of morpholinos in proximal tubule epithelial cells, highlighting their potential as a therapeutic platform for the treatment of renal disease.

Highlights

CYP3A5 is the primary CYP3A subfamily enzyme expressed in the human kidney and its aberrant expression may contribute to a broad spectrum of renal disorders
Incorrect splicing of exon 4 at the *3 single nucleotide polymorphism (SNP) site creates a pseudo-exon with a termination codon, located upstream of the exon 4 splice acceptor site, capable of triggering nonsense mediated decay (NMD) and loss of the CYP3A5 mRNA
To clarify the role of the *3-SNP in promoting NMD of mature transcripts, we explored the nature of CYP3A5 mRNA expression in a human embryonic kidney (HEK293) cell line, which was verified as homozygous *3/*3 by DNA sequencing (Supplemental Fig. 1b)

Summary

Introduction

CYP3A5 is the primary CYP3A subfamily enzyme expressed in the human kidney and its aberrant expression may contribute to a broad spectrum of renal disorders. Morpholinos targeting a cryptic splice acceptor created by the CYP3A5*3 mutation in intron 3 rescued functional CYP3A5 expression in vitro, and salt-sensitive cellular mechanisms regulating splicing and conditional expression of CYP3A5*3 transcripts are reported. We previously showed the utility of using ASO interference to alter spliceosome function by targeting an exon splice-acceptor site in the c-myc transcript[38] This strategy has been used to restore the reading frame in mutant forms of dystrophin; create a ligand independent CTLA-4 splice variant; and modulate immune responses to Ebolavirus[39,40,41,42,43,44]. The current studies utilized synthetic oligomers to confirm the role of the *3 SNP and a putative, intron 3 G-quadruplex in enhancing the exon 4 splice-acceptor in CYP3A5*3/*3, which leads to restoration of functional enzyme expression under high salt conditions. We demonstrate that CYP3A5 expression can alternatively be modulated using ASOs that interact with target RNA via Watson–Crick base p airing[49,50]

Methods

Results

Conclusion