Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria

Abstract

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

Efficient Synthesis of 2’‐O‐Methoxyethyl Oligonucleotide‐Cationic Peptide Conjugates

“…The retention times of the macromolecules was used as a measure of their lipophilicity, as previously reported. [23] Surprisingly, we observed only minor differences in the retention times of the conjugates and the parent 5’ maleimide oligonucleotide, but far from those observed after conjugation of the oligonucleotide to hydrophobic moieties such as stearic acid or cholesterol (Figure S3[ 19 , 23 ]).…”

“…[18] We examined oligonucleotide conjugation to either the N-terminus or the C-terminus for each peptide, since peptide orientation may impact the pharmacokinetic properties of the conjugate [11a] (Table 1). As a test sequence, we selected a 22-mer 2'-MOE-PS oligonucleotide that we have used previously to modulate splicing of the FECH pre-mRNA in the context of erythropoietic protoporphyria (EPP), [19] a rare disease caused by accumulation of the toxic heme precursor protoporphyrin IX in red blood cells. [20] We employed a solution phase thiol-maleimide conjugation protocol for the preparation of structurally diverse oligonucleotide conjugates.…”

Erythropoietic protoporphyria: time to prodrome, the warning signal to exit sun exposure without pain—a patient-reported outcome efficacy measure

“…Given the rarity of EPP, it is important to identify reliable and reproducible endpoints for clinical trials. 24 Since new therapies are being developed by several companies and academic laboratories, [25][26][27][28] it is important to evaluate new potential primary efficacy endpoints to allow assessment of a drug's safety and effectiveness, without risking prolonged sun exposure and incapacitating pain attacks. Therefore, we hypothesized that average daily TTP may be a more patient-compatible primary outcome measure to investigate the clinical efficacy of treatments for EPP.…”

RNA‐PROTACs: Degraders of RNA‐Binding Proteins

“…Here we describe RNA-PROTACs,anew class of chimeric oligonucleotides that are rationally designed to target RBPs,t hat is,p roteins.M any groups have conjugated oligonucleotides to peptides,usually however,for the peptide to transport the oligonucleotide to its target RNAi nv itro/ vivo. [45] Here,the roles of the peptide and the oligonucleotide are reversed;t he oligonucleotide delivers the peptide to its target site.R NA-PROTACsd ock into the RNAb inding site of an RBP via the structurally modified oligoribonucleotide that is sequence-identical with the native RNA-binding element of the RBP.T he first RNA-PROTACt argets the Lin28 protein, as tem cell factor and oncoprotein of high interest as ap otential drug target for several diseases.U sing as tructure-based approach, we designed a7 -nt PS-MOE oligonucleotide analogue that binds tightly to the zinc finger domain of Lin28A. AV HL-recruiting peptide that is conjugated to the 5'-end of the oligonucleotide then mediates degradation of the target in cells via the ubiquitination pathway.T his proof-of-concept represents an ew means of degrading and thereby inhibiting RNA-binding proteins, at arget class that until now has proven difficult to address pharmacologically.…”