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.
Main Text
Ferrochelatase (FECH[MIM: 612386]) is an inner mitochondrial membrane enzyme that catalyzes the insertion of ferrous iron into free protoporphyrin IX (PPIX) to form heme, required for the synthesis of various hemoproteins, including the cytochromes and hemoglobin. Erythropoietic protoporphyria (EPP [MIM: 177000]) is a rare inherited disease caused by a systemic deficiency in FECH activity; this activity is particularly critical in bone marrow erythroid cells, and its deficiency results in the accumulation of the FECH substrate PPIX. EPP is a lifelong disease characterized by painful photosensitivity; it is responsible for social disability and it has a pronounced impact on affected individuals’ quality of life. Approximately 2% of affected individuals develop advanced cholestatic liver disease and are at risk of rapidly developing fatal liver failure, requiring liver transplantation.1 In 90% of cases, the inheritance of EPP in individuals with the overt disease reveals the presence of an LOF mutation in trans to a common hypomorphic c.315−48C FECH allele (rs2272783).2 The hypomorphic c.315−48C allele decreases FECH activity as a result of the favored usage of a cryptic acceptor site in intron 3; use of this site leads to the production of a 63-bp-longer mRNA, the introduction of a premature stop codon, and subsequent degradation by nonsense-mediated mRNA decay (NMD)3 (Figures 1A and 1B). The consequent reduction in enzymatic activity, to approximately 35% of normal levels, is sufficient for symptoms to occur. Individuals with only an LOF allele show 50% of residual FECH activity and are asymptomatic.2 Based on these observations, the restoration of correct intron 3 splicing should be sufficient to restore a normal phenotype in individuals carrying this splice variant.
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.
We recently identified an antisense oligonucleotide, AON-V1 (V1), which restores end-to-end exon3-exon4 physiological splicing and rescues ex vivo a normal phenotype in erythroblasts of EPP-affected individuals.4 The objective is to develop a delivery strategy designed to target in vivo V1 to the right cell at the right time. Notably, critical steps are passage through the membranes of these cells, transport to and across the nuclear membrane, and interactions with FECH pre-mRNA. Among targeted delivery systems, non-viral gene carriers are safer than viral vectors in terms of controlled gene expression and lack of an immunogenic response. Unfortunately, because of the lack of a specific delivery system and the difficulty in transfecting erythroid progenitors, erythroid progenitor-targeted gene therapy mediated by non-viral agents has never been reported. As evidenced by liver-targeting methods, a cargo molecule targeting a natural receptor that is expressed at high levels on the cell type of interest (i.e., an asialo-glycoprotein receptor in the case of hepatocyte-targeting systems) dramatically improves the efficiency of transfection and reduces toxicity and off-target effects.5 Holo-transferrin (Holo-TF) binds with high affinity to transferrin receptor 1 (TFR1) and triggers endocytosis to import iron into all cells. Iron is delivered into the cell through the divalent metal transporter 1 (DMT1), and TFR1 is recycled back to the cell surface.6 Because of the requirement for large amounts of iron for heme synthesis and the maintenance of cellular survival, erythroid tissue expresses the highest levels of TFR1. During erythropoiesis, TFR1 expression begins to increase between the burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) stages,7 mainly increases during the terminal differentiation up to the late basophilic stage, and decreases at the orthochromatic stage.8 Interestingly, FECH expression is also strongly induced during terminal erythropoiesis so that iron can be incorporated into PPIX to form heme.
Because of the high concentration of TF in the blood, TF-conjugated delivery vectors show low transfection efficiency, highlighting the importance of targeting a receptor-binding site different from the TF site.9
Using TFR1 as a Trojan Horse to Address Antisense Oligonucleotide to Erythroid Cells
In this study, we designed bifunctional peptides comprising TFR1 ligand sequences conjugated to a modified Cys-(D form-R9)-Cys (9R) cell-penetrating peptide (CPP). The positive charge of the 9R sequence allows non-covalent complexation with negatively charged oligonucleotides (Figure 1C). This strategy has the advantage of avoiding laborious chemical conjugation of the CPP with its cargo and ultimately lowers the AON concentrations that are generally required to achieve a biological response.9
Selection of the Most Efficient and Least Toxic P-9R/V1 Nanocomplex, Which Corrects Splicing in Lymphoblastoid Cell Line (LBLC), from an Overt EPP Subject
On the basis of the literature describing peptides that interact tightly with TFR1, we selected five peptides and conjugated them to 9R CPP (Table S2). We used LBCLs from an overt EPP subject (subject II1, FECH genotype: c.709delT mutation/c.315-48C, Figure 1D) and her asymptomatic brother (subject II3, FECH genotype: c.709delT mutation/c.315-48T, Figure 1D) to investigate the effects of individual P-9R/V1 nanocomplexes on splicing. Subject II1 had severe photosensitivity and underwent two liver transplantations due to EPP liver dysfunction. Unfortunately, the individual died as a result of liver failure at 48 years (25 years after the first liver transplantation). Among the above-mentioned five identified peptides, we selected the most efficient, named P1-9R. The latter and P2-9R showed the best efficiency at restoring intron 3 splicing at the lowest relative charge ratio between P-9R and V1 (5:1 for P1-9R, Figures 2 and S1). We selected the shorter peptide (P1) to reduce potential immunogenicity, and then we characterized this peptide more extensively. A time-course experiment performed in II1 LBCLs transfected with the P1-9R/V1 nanocomplex (charge ratio 5:1) showed a prolonged effect that persisted for at least 96 h after transfection (Figure 3). This persistence suggests a protective effect of the nanocomplex on V1 against serum nuclease degradation and/or the persistence of V1 in the cytoplasm.
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.
The hydrodynamic size of the P1-9R/V1 nanocomplex in different media is less than 200 nm, which limits the risk of its rapid opsonization by the mononuclear phagocyte system,10 and is greater than 5 nm, which limits the risk of rapid renal clearance and urinary excretion11 (Table S3A). Zeta-potential measurements obtained in different media showed a transition from a negative charge to a positive charge as the relative-charge ratio of peptide to V1 increased (Table S3B). Based on these data, in vivo administration of the P1-9R/V1 nanocomplex would require an increase in the charge ratio to reduce the risk of toxicity because a higher zeta potential indicates a more stable nanocomplex (Table S3B). 72 h after the transfection of II1 LBCLs with the P1-9R/V1 nanocomplex (charge ratio 5:1), cell viability was determined via a standard MTT assay, and no evidence of cytotoxicity was detected (Figure S2). Finally, we observed that the plasma from a normal and an EPP subject had no impact on splicing correction by P1-9R/V1. This suggests that V1 is protected from plasma endonucleases by its complexation with P1-9R (Figure S3).
Holo-TF Concentration Has a Limited Effect on P1-9R/V1 Nanocomplex Endocytosis and Splicing Correction
Iron availability is the main determinant of TFR1 expression through the IRE (iron responsive element) and IRP (iron regulatory protein) regulation of TFR1 mRNA stability. The erythroid UT7-EPO (EPO: erythropoietin) cell line, established from a Japanese individual, is heterozygous c.315−48C/T at the FECH locus. To investigate whether iron availability could modulate P1-9R/V1 nanocomplex endocytosis and finally could impact the efficiency of splicing correction, we applied various Holo-TF concentrations, ranging from 0 to 1,000 μg/mL, to the UT7-EPO cell culture media. As anticipated, the absence of Holo-TF in the media dramatically increased TFR1 expression (Figure S4A) and improved the efficacy of the P1-9R/V1 nanocomplex to correct splicing (Figure S4B; intron3-exon4 cryptic splicing is almost undetectable). Nevertheless, whatever the other Holo-TF concentrations, including the maximum dose of 1,000μg/mL, splicing correction was efficiently obtained (Figure S4B, 6–9% of residual cryptic splicing versus 36–42% in the mock condition) and was at the same level as in cells with the wild-type c.315-48T/T FECH genotype.4
The P1-9R/V1 Nanocomplex Uses the TF/TFR1 Endocytosis-Cycle Internalization Pathway
We starved human fibroblasts of fetal bovine serum (FBS) and treated them with P1-9RFITC or P1RhodamineB concomitantly with Alexa Fluor 647-labelled TF (TFAlexaFluor 647) to monitor the intracellular trafficking of the P1-9R peptide. All peptides were detected in the cytoplasm within 30 min. Confocal micrographs verified the lack of artefacts in the internalization pattern. The co-localization of TFAlexaFluor 647 with either P1-9RFITC or P1Rhodamine B peptides was mainly observed in the cytoplasm. Thus, internalization of the P1-9R or P1 peptides utilized the TF/TFR1 endocytosis-cycle internalization pathway. On the other hand, the P1-9R peptide entered the cells more efficiently than P1 alone and completely co-localized with TF at the cell membrane and in the cytoplasm (Figure S5). We concluded that the P1 peptide conjugated to 9R was internalized by the TFR1/TF pathway, in addition to other possible pathways specific to 9R.12
Altogether, the P1-9R/V1 nanocomplex showed good bioavailability and an optimal size and did not induce toxicity. Therefore, the nanocomplex was used at a 5:1 charge ratio in subsequent experiments.
The P1-9R/V1 Nanocomplex Corrects Splicing and Increases FECH mRNA and Protein in LBCLs from an Overt EPP Subject
We further investigated whether the restoration of normal FECH mRNA maturation (Figure 4A) also increased WT FECH mRNA production. After the transfection of II1 LBCLs with the P1-9R/V1 nanocomplex, we measured amounts of the WT FECH mRNA by performing RT-qPCR with primers specific for the WT exon3-exon4 boundary. As expected, a similar level of normal mRNA was observed in the cells from subject EPP II1 treated with P1-9R/V1 and the cells from asymptomatic subject EPP II3, and the amount of mRNA increased by 30% relative to those of II1 LBCLs that were untreated or treated with P1-9R/Mock (Figure 4B). In contrast to P1-9R/V1, P2-9R/V1 was unable to statistically significantly increase WT FECH mRNA production and therefore was not used in subsequent experiments (data not shown). Importantly, 72 h after transfection, FECH immunoblotting revealed a correlation between WT FECH mRNA production and an increase in FECH amounts in P1-9R/V1-treated II1 LBCLs relative to P1-9R/Mock-treated or untreated cells (P1-9R/V1: +133% relative to untreated cells and +116% relative to P1-9R/Mock-treated cells, Figure 4C). Finally, FECH expression in II1 LBCLs treated with P1-9R/V1 was restored to the same level as that subject II3, subject II1’s asymptomatic brother (Figure 4C).
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.
The P1-9R/V1 Nanocomplex Corrects Splicing, Increases FECH mRNA, and Reduces PPIX Overproduction in Primary Cultures of CD34+ Progenitors from an Overt EPP Subject
Because medullary erythroblasts are the relevant tissue that is therapeutically targeted in EPP, we tested ex vivo the ability of the P1-9R/V1 nanocomplex to differentiate CD34+-derived erythroid progenitors isolated from the peripheral whole blood of an overt EPP subject and a control subject (see Supplemental Note). A culture of CD34+ cells isolated from a non-porphyria subject carrying a c.315-48C hypomorphic allele was first established so that the optimal conditions for nanocomplex treatment could be determined. The time course of splicing correction was monitored by FECH mRNA exon3-exon4 RT-PCR. After a single transfection with P1-9R/V1 at D7, splicing correction was maintained for at least 96 h (Figure S6).
To differentiate CD34+ cells from the EPP subject, we performed transfection with the P1-9R/V1 nanocomplex at the intermediary erythroblast stage (CD34low, CD36high, CD71high, GPAhigh, mainly basophilic erythroblasts), when the FECH enzyme is expressed at high levels concomitantly with TFR1. This stage corresponded to D9 post-CD34+ cell isolation and culture (data not shown). Two days after transfection (D11), cell proliferation and morphology were identical after May-Grünwald-Giemsa (MGG) staining, regardless of treatment (no treatment or transfection with P1-9R/Mock or P1-9R/V1) and showed no evidence of toxicity (Figures S7 and S8). 24 h after transfection, emetine was added to the medium at a final concentration of 30 μM, and splicing correction was analyzed by exon3-exon4 RT-PCR of the FECH mRNA at D12 (72 h post-transfection). Intron 3 retention was almost completely corrected in cells treated with the P1-9R/V1 nanocomplex (Figure 5A; expressed as the ratio of the aberrantly spliced RNA to total RNA; NT [non-treated]: 1; P1-9R/Mock-transfected cells ratio: 1.02; P1-9R/V1-transfected cells ratio: 0.12). For WT FECH mRNA quantification, cells that were not treated with emetine were also harvested 72 h (D12) and 96 h (D13) after transfection. Compared to untreated cells, cells transfected with P1-9R/V1 had significantly increased WT FECH RNA expression 72 h after transfection (P1-9R/V1: +13% relative to untreated cells; Figure 5B). 96 h after transfection, only P1-9R/V1-treated cells showed a significant increase in WT FECH mRNA amounts relative to amounts in untreated and P1-9R/Mock-treated cells (P1-9R/V1: +18% relative to untreated cells; Figure 5B). Finally, PPIX accumulation in erythrocytes was analyzed by flow cytometry with the violet laser of the FORTESSA flow cytometer (BD Biosciences, PPIX excitation: 405 nm, emission filter: 610/20 nm). At 72 and 96 h after transfection, the level of PPIX accumulation was significantly decreased in cells transfected with P1-9R/V1 (72 h: −19% relative to untreated cells or P1-9R/Mock-treated cells; 96 h: −17%; Figure 5C).
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.
Our study provides a proof-of-concept for the ability of TFR1 ligand-peptide coupled to 9R CPP to efficiently target V1 to developing erythroblasts to restore normal exon3-exon4 pre-mRNA splicing and increase normal FECH mRNA production while decreasing PPIX accumulation in cultured erythropoietic cells from an individual with EPP. Currently, an etiopathogenic treatment for EPP is not available. The only curative treatments are allogenic bone-marrow transplantation or either integrative or genome-editing gene therapy. Because of the associated high morbidity and mortality, bone-marrow allografts are restricted to those who have undergone a liver transplant, so that the allografts might prevent damage to the transplanted liver. Additive gene therapy is currently designed for individuals with severe hematological genetic diseases, and therapeutic genome editing is a prospective option. Finally, interest in non-integrative AON gene therapy is highlighted by (1) the need for a modest improvement in FECH activity in erythroblasts (when FECH activity is compared in individuals with overt and asymptomatic EPP), (2) the presence of the c.315-48C allele in more than 90% of EPP-affected individuals, (3) the approximately 120-day lifespan of circulating erythrocytes, suggesting prolonged effects of AON treatments, and (4) the seasonality of EPP. The development of AON treatments for genetic diseases is experiencing considerable growth. Two drugs have received marketing authorization in the USA: Nurinersen for spinal muscular atrophy12 and Eteplirsen for Duchenne muscular dystrophy.13, 14 Their use is nevertheless limited either by an intrathecal invasive administration route for Nurinersen or by an overall modest therapeutic effect for Eteplirsen. Despite advances in AON chemistry and design, systemic use of AONs is limited by poor tissue uptake, and thus sufficient therapeutic efficacy is difficult to achieve. The objective of this approach remains the in vivo targeting of the therapeutic AON at the right time to the right cell, i.e., the erythroblasts in EPP. Given that AON has a short period of stability in the serum as a result of nuclease degradation and urinary elimination of small hydrophilic molecules, successful erythroid-targeted AON transport also requires formulations that completely condense AON into nanoparticles in vitro and remain stable in vivo. Targeted delivery vehicles that deposit a bioactive therapeutic AON would also reduce undue toxicity. The use of an artificial ligand specific for a membrane receptor (also called the Trojan horse approach) enables the efficient delivery of a therapeutic DNA to a given tissue. For example, the use of trivalent N-acetylgalactosamine (GalNAc), which binds with high affinity to the asialoglycoprotein receptor expressed on hepatocytes, enables the efficient and non-toxic delivery of RNAi therapeutics to the liver and is currently used in clinical studies.15 One strategy for increasing V1 bioavailability in erythroblasts and decreasing its toxicity towards other tissues would be to utilize artificial ligands targeting TFR1. TFR1 is expressed at high levels on developing erythroblasts (400,000 to 800,000 molecules per cell),6 displays a high rate of endocytosis and turnover (20,000 molecules/cell/minute16) and is expressed concomitantly with FECH. The transfection efficiency of transferrin-conjugated delivery vectors is low as a result of competition with the high concentrations of transferrin (25 μM) in the circulation.17 Transferrin vectors also impair iron metabolism and TFR2 functions. Therefore, strategies targeting TFR1 through the use of specific TFR1 ligand-peptides may help circumvent these concerns. The potential problem with the manipulation of TF uptake as a means of drug delivery across cells is the TFR1 recycling pathway18 and the necessity for the AON to exit the endosome to reach the nucleus. We coupled the TFR1 ligand-peptide to a 9R CPP composed of Cys-(D-R9)-Cys peptide units linked together by disulfide bridges to improve exosomal escape of V1.19 This method enabled the stable formation of 200 nm nanocomplexes through electrostatic interactions between the negatively charged AON and the cationic oligomer-peptide complex. Once in the endosome, the disulfide bridges of the polypeptide are reduced, and thus the nucleic acid is more readily released into the cytosol so that it can enter the nucleus.
Altogether, the TFR1 ligand-peptide coupled to the 9R peptide appears to be a promising tool for targeting AONs and, broadly, DNA to developing erythroblasts, which represent one of the most common cells implicated in genetic diseases, such as hemoglobin disorders. Testing this strategy in a humanized mouse model of EPP would pave the way for future treatments of EPP in humans.
Declaration of Interests
The authors declare no competing interests.
Acknowledgements
We thank the affected individuals who kindly contributed to this study. We thank Alain Martelli for his generous gift of the FECH antibody and Anne-Marie Robreau and Nicolas Ducrot for providing excellent technical assistance. This study was supported by a grant from Laboratory of Excellence GR-Ex, reference ANR-11-LABX-0051. A.M. was supported by a Labex GR-Ex scholarship. The Labex GR-Ex is funded by the program “Investissements d’avenir” of the French National Research Agency, reference ANR-11-IDEX-0005-02.
Published: January 31, 2019
Footnotes
Supplemental Data include a Supplemental Note, Supplemental Results, eight figures, four tables, Supplemental Methods, and Supplemental References and can be found with this article online at https://doi.org/10.1016/j.ajhg.2018.12.021.
Web Resources
GTEXPortal, https://www.gtexportal.org/home/eqtls/byGene?geneId=FECH&tissueName=All
Online Mendelian Inheritance in Man, http://www.omim.org
Supplemental Data
References
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