Erythroid-Progenitor-Targeted Gene Therapy Using Bifunctional TFR1 Ligand-Peptides in Human Erythropoietic Protoporphyria

Abstract

Erythropoietic protoporphyria (EPP) is a hereditary disease characterized by a deficiency in ferrochelatase (FECH) activity. FECH activity is responsible for the accumulation of protoporphyrin IX (PPIX). Without etiopathogenic treatment, EPP manifests as severe photosensitivity. 95% of affected individuals present a hypomorphic FECH allele trans to a loss-of-function (LOF) FECH mutation, resulting in a reduction in FECH activity in erythroblasts below a critical threshold. The hypomorphic allele promotes the use of a cryptic acceptor splice site, generating an aberrant FECH mRNA, which is responsible for the reduced level of wild-type FECH mRNA and, ultimately, FECH activity. We have previously identified an antisense oligonucleotide (AON), AON-V1 (V1), that redirects splicing to the physiological acceptor site and reduces the accumulation of PPIX. Here, we developed a specific strategy that uses transferrin receptor 1 (TRF1) as a Trojan horse to deliver V1 to erythroid progenitors. We designed a bifunctional peptide (P₁-9R) including a TFR1-targeting peptide coupled to a nine-arginine cell-penetrating peptide (CPP) that facilitates the release of the AON from TFR1 in endosomal vesicles. We demonstrated that the P₁-9R/V1 nanocomplex promotes the efficient and prolonged redirection of splicing towards the physiological splice site and subsequent normalization of WT FECH mRNA and protein levels. Finally, the P₁-9R/V1 nanocomplex increases WT FECH mRNA production and significantly decreases PPIX accumulation in primary cultures of differentiating erythroid progenitors from an overt EPP-affected individual. P₁-9R is a method designed to target erythroid progenitors and represents a potentially powerful tool for the in vivo delivery of therapeutic DNA in many erythroid disorders.

Figure 1

Molecular Mechanism and Targeting Strategy (A) Schematic representation of exon3-exon4 splicing of the FECH mRNA. The c.315−48T>C transition (rs2272783) modulates the splicing efficiency by promoting the use of a constitutive cryptic acceptor splice site. −63 bp: position of the cryptic acceptor splice site. The use of a cryptic acceptor site in intron 3 leads to the production of a 63-bp-longer mRNA, the introduction of a premature stop codon, and degradation by the NMD mechanism. (B) Cis-eQTLs for FECH in 19 different tissue types (50 kb window) from the GTEx Project portal. Bubble size represents −log10 (p value), and color and shading of the bubble represent the effect size of the cis-eQTL. TSS: transcription start site. rs2272783 appears as the most significant cis-eQTL (p = 1.2×10⁻²⁰ in fibroblasts), with a strong effect size (ES: −0.63, ancestral T allele relative to the derived C allele), and it appears in the largest number of tissues. rs2269219 (alias IVS1-23C/T) has been proposed to contribute to the low-expression mechanism. It shows a smaller and less significant effect (ES: −0.23; p = 1.7 × 10⁻⁷) in only one tissue as a result of partial linkage disequilibrium with rs2272783 (r² = 0.272). (C) Strategy used to deliver V1 to erythroid progenitors. The bi-functional peptides are composed of two parts: a TFR1-targeting part and a CPP part. The negative charges of V1 allow it to participate in electrostatic interactions with positively charged CPP (step 1). Once the CPP-TFR1/V1 nanocomplex (step 2) is formed, it is internalized in cells through TFR1 endocytosis (step 3). Because of the endosomolytic characteristics of the bi-functional peptide, V1 escapes from the endosomal vesicle (step 4). Once the peptide enters the nucleus, V1 recognizes the FECH pre-messenger RNA and corrects the splicing anomaly (step 5). (D) Pedigree of the EPP-affected family. “M” indicates the c.709delT LOF FECH mutation. “T” indicates the c.315−48T allele. “C” indicates the c.315−48C allele. Subjects I1 and II3 were asymptomatic carriers of the c.709delT mutation (GeneBank: NM_000140.3; FECH genotype M/c.315−48T), and subjects II1 and II2 had overt EPP (FECH genotype M/c.315−48C). FC: FECH activity is indicated in nmol of Zn-mesoporphyrin/mg of protein/h.

Figure 2

Identification of the Most Efficient P₁-9R/V1 Charge Ratio to Repress intron3-exon4 Cryptic Splicing LBCLs from an overt EPP subject (subject II1) were transfected with the P₁-9R/V1 nanocomplex at charge ratios of 1:1, 3:1, 5:1, 7:1, and 10:1 (peptide:oligonucleotide; V1 final concentration 428 nM), the P₁-9R/Mock nanocomplex (5:1), or V1 without the peptide. NT: non-treated. Cells were treated with emetine 48 h after transfection (final concentration 30 μM) so that NMD would be blocked. Total RNA was extracted with the RNA Plus reagent 72 h after transfection. Exon3-exon4 FECH primers amplifying the correctly and aberrantly spliced mRNA molecules were used for end-point PCR. PCR products were analyzed by electrophoresis on a 3% agarose gel. The intensity of the amplicons was measured with ImageJ Software. The results were calculated as the ratio of the intensity of the aberrantly spliced band (192 bp) to the intensity of the correctly spliced band (128 bp) plus that of the aberrantly spliced band.

Figure 3

Kinetics of Splicing Correction by the P₁-9R/V1 Nanocomplex LBCLs from the EPP overt subject (subject II1) were transfected with P₁-9R/V1 or P₁-9R/Mock at a 5:1 charge ratio or with P1-9R without V1. 24 h prior to RNA extraction, cells were treated with emetine. Intron3 splicing was examined via FECH exon3-exon4 end-point RT PCR.

Figure 4

P₁-9R/V1 Restores FECH RNA Expression and FECH Protein Production in LBCLs from Individuals with EPP LBCLs from the EPP overt subject (subject II1) were transfected with P₁-9R/V1 or the P₁-9R/Mock nanocomplex at a 5:1 charge ratio, V1, or P₁-9R without AON. NT: non-treated. (A) Intron 3 splicing correction was examined via FECH exon3-exon4 end-point RT PCR after emetine addition at 48 h. Total RNA was extracted 72 h after transfection. (B) The WT FECH mRNA was quantified 72 h after transfection by RT-qPCR with primers specific for the WT exon3-exon4 boundary. The results from 14 independent experiments were analyzed with the Mann-Whitney test. The graphic shows the mean ± standard deviation. (C) Immunoblotting with anti-FECH and anti-GAPDH antibodies allowed the measurement of protein amounts 72 h after transfection. Band intensity was measured with ImageJ software, and FECH amounts were normalized to the GAPDH level.

Figure 5

Decreased Accumulation of PPIX in Developing Primary Erythroblasts of an Overt EPP Subject after Treatment with P₁-9R/V1 Differentiating CD34+ cells from an overt EPP subject were transfected with the P₁-9R/V1 nanocomplex at D9. The final concentration of V1 was 428 nM. The charge ratio of peptide to AON was 5:1. (A) 24 h after transfection, intron3 splicing correction was examined via FECH exon3-exon4 end-point RT PCR after emetine addition. The ratio of the aberrantly spliced RNA to total RNA is indicated. (B) Total RNA was extracted 72 or 96 h after transfection so that the restoration of WT FECH RNA amounts could be studied. The amount of WT FECH mRNA was analyzed by RT-qPCR with primers specific for the WT exon3-exon4 boundary. The results from six intra-replica experiments were analyzed with a Mann-Whitney test. The graphic shows the mean ± standard deviation. (C) Cells were collected 72 and 96 h after transfection, washed, and re-suspended in FACS buffer so that PPIX accumulation could be measured. PPIX accumulation was analyzed by flow cytometry with the violet laser of the Fortessa flow cytometer (BD Biosciences, PPIX excitation: 405 nm laser, emission filter: 610/20 nm). The results from six intra-replica experiments were analyzed with the Mann-Whitney test. The graphic shows the mean ± standard deviation.