Under normal erythropoiesis, erythroblasts contain ~200 mitochondria per cell, playing crucial role in iron metabolism and biosynthesis of heme and iron-sulphur [Fe-S] clusters [20]. More than 80 percent of body heme levels are synthesized in ErPCs (BFU-Es, CFU-Es) for the production of hemoglobin [21], which corresponds to ~ 40-50% of total protein in mature RBCs. Disruption of interconnection between heme biosynthesis, [Fe-S] biogenesis and iron - heme proteins could result in congenital anemias (CGAs) [20].

Sideroblastic anemias are either acquired (associated with drugs or alcohol abuse) or congenital. They are characterized by the appearance of ringed sideroblasts (erythroblasts characterized by the accumulation of non-heme iron in perinuclear mitochondria) as common feature in the BM [20,22].

Mutations in the erythroid-specific heme biosynthetic gene ALAS-2 (δ-aminolevulinic acid synthase - 2, isoform exclusively synthesized in ErPCs) can lead to X-linked sideroblastic anemia (XLSA) (OMIM: 300751), the most common CGA. More specifically, some mutations intervene with the binding of ALAS-2 to the succinyl-CoA synthetase that provides the succinyl-CoA, necessary for the synthesis of δ-aminolevulinic acid (ALA), the first enzyme in the biosynthesis of heme [23,24]. Patients carrying mutations in ALAS-2 that influence the affinity of the enzyme for its cofactor pyridoxal phosphate may respond to treatment with pyridoxine (vitamin B6) [25,26,27].

Sideroblastic anemias may also occur by mutations in genes encoding transporters, like: (a) the solute carrier SLC25A38, an erythroid specific transporter of glycine into the mitochondrial matrix (OMIM: 205950) [28,29], where with succinyl-CoA will give genesis to ALA; and (b) the inner mitochondrial membrane ABCB7 transporter [30]. This transporter controls the availability of mitochondrial iron to be inserted in the protoporphyrin IX via ferrochelatase (FECH) [25,29]. Mutations in ABCB7 transporter may disrupt the maturation of [Fe-S] [31] in FECH during heme biosynthesis and lead to X-linked sideroblastic anemia with ataxia (XLSA-A) (OMIM: 301310). There is also evidence that deficiency of glutaredoxin 5 (GLRX5), also essential for [Fe-S] biogenesis, can lead to sideroblastic anemia in humans (OMIM: 609588) [20,32].

Furthermore, congenital sideroblastic anemias are associated with abnormalities concerning mitochondrial protein synthesis, like: (a) Pearson Marrow-Pancreas Syndrome (PMPS) [33] (OMIM: 557000), with half patients being heteroplasmic for the 4977-bp common deletion in the mitochondrial genome; (b) Mitochondrial myopathy with Lactic Acidosis and ringed Sideroblasts (MLASA) [34] (OMIM: 600462), with mutations in PUS1 gene, encoding PUS playing role in the metabolism of uridine; and (c) Thiamine Responsive Megaloblastic Anemia (TRMA) [35] (OMIM: 249270) due to mutations in thiamine transporter SLC19A2. Unfortunately, there is no cure for congenital sideroblastic anemias up to now, while gene therapy is considered as a possible future therapeutic intervention [20].

“Porphyrias” represent a group of inherited or acquired disorders attributed to deficiencies of enzymes involved in heme biosynthesis. Porphyrin intermediates (substrates for the corresponding defective heme biosynthetic enzymes) accumulate in the BM erythropoietic cells and/or in the liver. Thus porphyrias are classified as erythropoietic or hepatic, respectively. Patients with porphyrias must avoid the use of the so called “porphyrogenic” drugs, which may enhance the frequency of acute porphyria attacks with serious complications [36,37].

More specifically, mutations in human heme biosynthetic genes can give genesis to porphyrias [38], like in: (i) ALAS-2, leading to X-linked dominant erythropoietic protoporphyria (XLEPP or XLDPP) (OMIM: 300752) [39,40]; (ii) UROS (uroporphyrinogen III synthase), leading to congenital erythropoietic porphyria (CEP) (OMIM: 263700) and (iii) FECH, leading to erythropoietic protoporphyria (EPP) (OMIM: 612386). Moreover, mutations in human GATA-1 can also result in CEP (OMIM: 305371) [41]. It is worth mentioning here that at least five heme biosynthetic enzymes have binding site for the GATA-1 (an erythroid specific transcription factor) [42].

Acute porphyria attacks are life threatening and can be manipulated by: a) avoiding precipitating factors [porhyrinogenic drugs (chloroquine, sulphonamide antibiotics, rifampicin), alcohol, hormones (estrogen, progesterone) that overproduce porphyrins)]; b) alleviating the neurological (nausea, vomiting, pain) or cardiovascular problems with symptomatic therapy; c) intravenous (i.v.) administration of hemin, in the form of heme arginate or hematin [38]. However, treatment with hemin should be monitored, since free heme accumulation could lead to iron overload and organ failure. Patients with chronic porphyrias (like EPP, CEP) present mild to severe cutaneous lesions (erythema, blistering and others) that exacerbated after expose to sunlight. Thus, in chronic porphyrias, intentional avoidance of sunlight is recommended. Liver or BM/HSC transplantations are recommended in severe cases [36,38,43].

Hemoglobinopathies [44], as inherited disorders of hemoglobin, are among the most common monogenetic ERDs. Hemoglobinopathies reflect the natural selection of carriers, being protected from the consequences of infection with P. falciparum that causes malaria [45,46,47,48]. A plethora of hemoglobinopathies, listed in the HbVardatabase [49], arise from globin gene mutations. About 5-7% of the world’s population carries globin gene variants, while ~340.000 annual births appear with major hemoglobin disorders [7,47,50].

CPP-mediated PTs are engineered via PTD Technology by: a) cloning of the CDS, corresponding to the open reading frame of the PT of interest, adjacent to the sequence encoding the CPP (like the TAT peptide) into a suitable expression vector (like the pTAT/TAT-HA from Dowdy’s group); b) subsequently transformation of appropriate competent (bacterial) cells;and c) production of fusion proteins containing the CPP of choice linked to the protein of interest [81,98,99]. In cases that the produced CPP-mediated PT needs post-translational modifications in order to exert its biological actions, novel modified eukaryotic cloning-expression systems are employed [100], since bacteria fail to carry out post translational modifications (e.g. glycosylation) of the produced protein.

Chemical conjugation can be also applied between the CPPs and the cargo of interest in order to obtain the CPP - cargo, which will be able to deliver and, later on, release the cargo (e.g. the desired PT) intracellularly, depending on the environmental pH, temperature or protease [84,101,102,103,104]. The most widely used linker for conjugation is the disulphide bridge, permitting reduction of the CPP-cargo after penetration of the cell membrane, due to the high concentration of intracellular glutathione and subsequent release of the cargo. However, a recent study proposed that such a reduction could also happen at the level of cell surface [105]. Other linkers proposed for chemical conjugation of the PTs with the CPP of interest are the maleimide linker bond, sulphanyl (bi-functional) bond as well as phosphatydylethanolamine hydrogen bonding [95,106].

Protein transduction can be also approached in a non covalent manner by either premixing of the selected CPP with the desired cargo or by formulating the CPP - cargo in liposomes or nanoparticles [107,108,109]. Non covalent molecular interactions between the CPPs and the cargo seems to depend on their tertiary structures and hydrophobic as well as electrostatic interactions [107].

PTD Technology, compared to gene therapy, is regarded safe, since it doesn’t affect/alter the host genome integrity [17,18,19], although obstacles remain to be solved. These obstacles include: (i) lack of tissue specificity; (ii) protein instability, induced by proteases being present in circulation and inside the cells; (iii) immunogenicity; and (iv) other adverse reactions (like hypersensitivity) of the delivered CPP-mediated PTs (Table 2).

Most of the CPPs applied for pharmacokinetic studies undergo rapid clearance from the blood, with liver and kidney considered as the primary organs of their accumulation [118]. The necessity for multiple times administration of CPP-mediated PTs at high doses may indeed increase their immunogenicity [117]. Stability of CPP-mediated PTs could be affected by exchanging the L-amino acids with the corresponding D-amino acids, although this procedure may reduce the intracellular delivery [113].

Polymerization technologies (e.g. protein PEGylation) as well as entrapment of proteins in liposomes and/or nanoparticles could achieve better pharmacokinetic profile for the CPP-mediated PTs and overcome a number of obstacles, allowing penetration of even intact biological barriers, like BBB or BSCB (blood-spinal cord barrier) [114].

The ability of CPPs to penetrate almost all biological membranes makes the targeted delivery of proteins a very important issue in order to minimize undesirable effects. One should take in consideration the special characteristics of the targeted organ microenvironment. Torchillin’s group [95,104] developed “smart” delivery platforms for organ-tissue specific targeting, after administration of the desired cargo (e.g. the PT of interest) in the circulation: CPP - cargo is shielded with a protective molecule, like PEG (polyethylene glycol polymer), thus enhancing solubility, decreasing accessibility for the circulating proteolytic enzymes and increasing the half time of the PT by reducing kidney filtration due to its increased size. PEG chains can carry target-specific antibodies, attached to the surface via bonds sensitive to cell environmental (e.g. pH, temperature, matrix metalloproteinases) or external (magnetic field, ultrasound) stimulus conditions. After accumulation of the “smart” nanocarrier in the targeted organ and release of the PEG-antibody conjugates, the exposed CPP permits the transduction of the cargo intracellularly. Such an approach seems reasonable for the design of tumor-specific drugs/prodrugs/biodrugs, although the observed cell heterogeneity in tumors raises a number of limitations.

Another interesting approach for targeting delivery could be the ability to attach, during the production process, a homing peptide, if any available, adjacent to the sequence of the suitable CPP-mediated PT of interest [110]. Such homing peptides, able to bind specific molecules on the targeted cell population and facilitate selective transduction of PT, could be identified by Phage Display technology [111]. The design of a CPP, specific for glial cells, has been presented as a promising therapeutic approach for glial-related disorders [112].

In cases of subcellular organelle-targeted therapy, specific targeted sequences [like NLS (nuclear localization signal) or MTS (mitochondrial targeting signal)]) can be inserted in the designed sequence (e.g. CPP-MTS-PT) [18,99,119,120,121]. In an in vivo mouse (E3-deficient) model, intramitochondrial delivery of lipoamide dehydrogenase (LAD; E3), fused to TAT peptide, restored LAD activity [122]. Another class of CPPs, the cationic polyproline helices (CAPHs), has been also proposed to reach even more efficiently the mitochondria via direct transport mechanism [91].

CPPs have been administered by various ways, both parentally as well as topically. Parental administration is the most frequent route for the delivery of biodrugs [107,110]. Parental administration is the most frequent route for the delivery of biodrugs. Oral delivery would open new opportunities in biodrugs’ administration, since it is non invasive and friendly to the patient for self administration, but it is not as yet applicable. The lack of specificity of CPP-mediated therapeutics towards various tissues till now led topical applications to be more preferable for clinical purposes [118]. Thus, CPP-mediated therapeutics (like PsorBan, RT-001, AZX-100), that have reached clinical trials, are mainly for local use towards scarring, psoriasis, pain, wrinkles and excessive sweating (see Table 3) [74,96,123,124]. Data from Phases I/II on more than 2,000 patients indicated no significant adverse reactions [125]. There are also clinical trials targeting heart problems or even cancer (Table 3). The so-called “smart” delivery platforms, bypassing stability and tissue specificity problems, would further facilitate the development of novel therapeutic interventions [95].

It has been quite clear that ERDs, described thus far, include diseases, where ErPCs or even mature RBCs are deficient in a gene or its translation product and thus fail to function properly. Therefore, it is reasonable to assume that ERT/PRT approach would be beneficial for these disorders. An enzyme or any other type of missing protein can be produced as recombinant protein outside the body in a selected gene cloning-expression system, modified accordingly via PTD technology to carry an appropriate CPP and then be transduced into ErPCs or RBCs (wherever is needed) to deliver its intended function.

This task, of course, requires careful study of each particular case of ERDs in order to design the appropriate experimental approach. A number of questions to be considered include: (i) what is the pathobiology of a given ERD; (ii) what type of ErPCs are involved; (iii) how are they characterized in terms of their cell surface antigens and progeny; (iv) what is the missing protein and its corresponding gene; (v) where is this protein located inside the cells; (vi) how does this protein function; and (vii) how shall this protein (PT) of interest be delivered and remain functional (in the form of a newly produced recombinant CPP-mediated PT) for relatively long time.This valuable information is considered essential prior to any design of experiments in order to clone, express and produce the CPP-mediated PT of interest in a highly purified state, biologically active and able to exert its function upon delivery into the target cells. A critical step for the successful treatment of ERDs by protein transduction is the isolation of the candidate recipient cells (target cells), deficient in the protein of interest, prior to transduction experiments. Current technologies, including flow cytometry and cell sorting complemented by functional assays, will be able to facilitate isolation of these target cells. These cells can be removed from the peripheral blood of patients, mobilizated by G-CSF, as it has been followed for HSC-based gene therapy for beta thalassemia [134]. Following characterization, isolated cells will be grown in culture at selective media with minimal manipulation and then be efficiently transduced with the CPP-mediated PT of interest for as long as needed. By the end of loading period, cells will be assessed for the efficiency of protein transduction and functionality, using conventional assays. Stability and biological activity of the transduced CPP-mediated PT are essential to correct the corresponding cellular protein deficiency and allow cells to act properly. Whether the transduced CPP-mediated PT will survive for as long as needed to correct the deficiency in the recipient cells is an issue for investigation. It may be possible for such cells to be submitted to repeated protein transductions in timed intervals. For example, ErPCs and/or RBCs, removed from patients, could be transduced ex vivo with the CPP-mediated PT of interest. It has to be tested whether transduction will affect the structural integrity and function of ErPCs and/or RBCs, causing hemolysis or other adverse effects. Furthermore, the transduced CPP-mediated PT is expected to enter the intracellular space and interact properly with other proteins or form functional protein complexes. Full comparability studies using deficient and transduced erythropoietic cells will be carried out in parallel to confirm that protein transduction delivers the intended therapeutic action. Successfully transduced, with the CPP-mediated PT, cells will then be administered back into the patients. In ERDs with aberrant protein located in the mitochondria, like in cases of mitochondrial enzymes ALAS-2 and FECH in X-linked sideroblastic anemia, X-lnked erythropoietic porphyria or erythropoietic protoporphyria, intramitochondrial targeted protein therapy via PTD technology should be applied [18,119].

Possible therapy of ERDs with the use of CPP-mediated PTs is still in its conceptual stage. At present, efforts can be made: (a) to produce erythroid related proteins (e.g. enzymes or oxygen carrier proteins) as fusion recombinant proteins bearing a CPP; and (b) to transduce them into either HSCs and/or ErPCs - RBCs. Basic studies on protein intracellular delivery, followed by preclinical studies with the use of relevant animal models, are needed to be evaluated, before clinical trials aimed to treat ERDs via CPP-mediated PTs would follow in the future. It is interesting, however, to underline that the field of CPP-mediated therapeutics (biodrugs) keeps growing in other areas of pathological disorders. Table 3 illustrates animal and human studies carried out or being on progress to assess the impact of the PTD technology in the treatment of such disorders. Of course, the roadmap of the development and application of PTD technology “from bench to bed” in people with ERDs is quite long, but is provocative and can be eventually fruitful.