Modelling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice

Abstract

Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3′ splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing.

Porphyrin-Induced Protein Oxidation and Aggregation as a Mechanism of Porphyria-Associated Cell Injury

“…Numerous genetic mouse models of porphyria have been described in which a particular gene in the porphyrin biosynthetic pathway is deleted/mutated, causing a blockade of the pathway and accumulation of porphyrin precursors proximal to the alteration. These include models of AIP, 17 congenital erythropoietic porphyria (CEP),18, 19 PCT, 20 HCP, 21 VP, 22 and EPP (ie, all except XLP and ADP) 23, 24. Many of these models phenocopy the pleiotropic features of human disease.…”

“…Although mouse models are beneficial, limitations exist in terms of mimicking the pathologic nature of disease in patients. For example, several EPP models—m1Pas FECH mutant,23, 34 FECH exon 10 deletion35, 36—provided valuable insights toward understanding the pathophysiology of EPP, but they do not fully mimic the commonly found (∼97% of all FECH-related EPP cases) c.315-48C polymorphism 24, 37. Given the high incidence of the c.315-48C polymorphism, it is a common therapeutic target for EPP 38, 39.…”

“…Given the high incidence of the c.315-48C polymorphism, it is a common therapeutic target for EPP 38, 39. To address the unmet need for a suitable preclinical EPP model, a humanized mouse EPP model carrying c.315-48C polymorphism has been developed 24 . Though limitations still exist, mouse models are important for preclinical studies including hepatocyte transplantation 40 and gene transfer 41, 42, 43…”

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

“…We elected to use MOE-PS SSOs because of their successful use in clinical programs (www.ionispharma.com) (33), their excellent safety record (33,34), their efficient distribution in vivo (16), the ease with which they can be elaborated with targeting groups or stereopure linkages (29). We employed a mini-gene assay transiently expressing the FECH-C variant in COS-7 cells, which we have described previously (21). For each SSO, we calculated the fraction of "correct" (desired) transcripts on agarose gels after work-up using RT-PCR.…”

“…We recently developed a transgenic mouse model for human EPP (Emi mouse) (21), incorporating the c.315-63 cryptic splice site of FECH from human intron 3. The mouse displays the aberrant splicing of exon 4; however, for reasons unknown, exon 3 is deleted from most transcripts ( Fig.…”