The term “inborn error of metabolism (IEM)” was firstly presented by Sir Archibald E. Garrod, in the 1908 during his studies on alkaptonuria (1). Since then, the number of diseases caused by IEM has increased, due to new identification techniques for the various biochemical phenotypes, and, since then, more than five hundred different IEM have been identified (2).
IEM cause inherited metabolic disorders (IMD) and classically they result from the lack of activity of one or more specific enzymes or defects in the transportation of proteins. The consequences can usually be the accumulation of substances present in small amounts, the deficiency of critical intermediary products, the deficiency of specific final products or the toxic excess of products of alternative metabolic pathways (3). The differences in the phenotypic manifestations observed depend on the severity of the affectation observed in the gene, the type and function of the enzyme affected, post translation mechanisms, cellular processes, genes in other loci that may also be affected and environmental factors. For all these factors, patients with IMD present a complex assembly of symptoms (2).
IMDs are considered rare disorders and according to the European Organization for Rare Disease (EURODIS), a disease is considered rare when 1 in every 2,000 inhabitants is affected. About 6,000 to 8,000 rare disorders are described and although the individual frequencies are low, collectively IMD represents around 6 to 8% of the EU population (4).
IMDs are inherited in a monogenic or Mendelian form, since one gene plays a predominant role in the determination of disease. The majority of IMDs (67%) are of autosomal recessive inheritance, 21% are of autosomal dominant inheritance, while 6% are X-linked and another 6% are related with defects in mitochondrial genome. In general, a high heterogeneity is observed and the kind and intensity of clinical manifestations are related to the type of mutations detected (5).
As monogenic disorders, IMDs gene mutations can be classified into four major groups: missense, nonsense, splicing and frameshift. Missense mutations are typically single nucleotide changes that either alter the amino acid in translated proteins or do not alter the amino acid. Nonsense mutations are point mutations in a sequence that create a premature stop codon (UAA, UAG or UGA) in the coding region of the mRNA, resulting in premature translation termination and, usually, a non-functional or rapidly degraded protein. Splicing mutations result in disruption of critical sequences for splicing and abolishment of the usual splice sites, or creation of aberrant or cryptic splice sites, which in turn resulting in aberrant proteins. Finally, frameshift mutations are commonly caused by deletion or insertion of a number of nucleotides that alter the reading frame for any subsequent downstream codons (6).
According to the pathophysiology, IMDs can be divided into three main categories; (i) disorders that give rise to intoxication, (ii) disorders involving energy metabolism and (iii) disorders involving complex molecules (7). Part of the first group are inborn errors of intermediary metabolism characterized by an acute or progressive intoxication due to a metabolic block and accumulation of toxic compounds, which includes inborn errors of amino acid catabolism such as phenylketonuria (7).
In terms of therapy, the main goal in a IMD is to re-establish the metabolic balance and for that, many strategies can be applied either isolated or in combination. The first approach is to either decrease the accumulating substrate with diet restriction or to inhibit the enzymatic activity. Another common strategies are the fast elimination of the toxic products from the body, an increase of the residual enzymatic activity (e.g. by co-factor administration) and the administration of the reaction end product in shortage (8).
Considering IMD medical care, the treatment represents a significant challenge to the public system, even with the scientific achievements of the last years. Until now, the majority of the available therapies are not definitive and only ameliorate the patient’s symptoms. For these reasons, several novel therapeutic strategies are arising and being largely investigated. They can be divided into two main groups: mutation specific therapies and non-mutation specific therapies.
1. Mutation Specific Therapy
The existence of a common type of mutations prompted the hypothesis of common type-specific molecular pathogenesis, in other words, nonsense, splicing and frameshift mutations usually lead to loss of or unstable protein whereas missense and in-frame insertion or deletion mutations produce nonfunctional or partially functional proteins. The comprehension of genotype-phenotype relationship has also led to the development of strategies based in the mutations nature, so that a therapeutic strategy developed against a certain type of mutation will be effective against similar mutations, regardless the gene (6,9).
1.1. Premature Termination Codon Read-through Therapy
Approximately 1800 inherited human diseases are caused by nonsense mutations (10). Suppressing, by reading through, the resulting premature termination codons (PTC) with compounds, allowing translation to continue to the true end of the transcript, is a promising approach for correcting this type of mutations. The read-through compounds reduce ribosome termination at the PTC, resulting in the insertion of a random amino acid and the translation of the remainder of the correct full length protein (11). PTCs that occur more than 50 nucleotides upstream of the final exon-intron junction generally induce transcript degradation through the nonsense-mediated mRNA decay suveillance pathway (12), which make any nonsense mutation that does not trigger significant nonsense-mediated mRNA decay a good candidate for this approach.
One of the PTC read-through compounds are aminoglycoside antibiotics. They bind to the decoding site of the 16S ribosomal RNA, inducing a local conformational change that allows translation through what would otherwise be read as a PTC (13). Paromomycin and G418 in mammalian cells (13) and gentamicin in clinical trials for cystic fibrosis, Duchenne muscular dystrophy and hemophilia (14,15) are aminoglycosides that have demonstrated to partially restore the full-length protein of nonsense mutation. However, for an effective read-through a high concentration of aminoglycosides is needed which is often toxic to cells and consequently restricts their uses in clinical trials. Furthermore, most antibiotics do not cross the blood-brain barrier efficiently and would be of limited use for treating central nervous system diseases. To reduce aminoglycosides’ cell toxicity while retaining the read-through activity, some attempts have been made in redesign their structure (16).
PTC124, a nonsense-suppression read-through compound recently identified, that is not an aminoglycoside antibiotic, was systemic delivered into mouse models for Duchnne muscular dystrophy and cystic fibrosis and showed restoration of protein and function in vivo without any obvious toxicity (17). PTC124 has demonstrated specific read-through activity to the PTC rather than to the true termination codons, promoting selective and specific read-through of disease-causing premature stop codons. However, before selecting PTC124 for the clinic, more than 800 000 chemicals are needed to be screened and then chemically modified (17). Du L et al., have identified more than 50 non-aminoglycoside chemicals with read-through activity which may contribute to a rapid expansion of the discovery aspect of read-through chemicals (18).
1.2. Antisense Oligonucleotide Therapy
Antisense Oligonucleotide therapy is based on an antisense oligonucleotide (ASO) – a single-stranded deoxyribonucleotide (typically 20 bp in length) – that is complementary to the target mRNA (19). Hybridization of ASO to the target mRNA via Watson-Crick base pairing can result in specific inhibition of gene expression by various mechanisms, depending on the chemical make-up of the ASO and location of hybridization, resulting in reduced levels of translation of the target transcript. The ASO is not only a useful tool for studies of loss-of-gene function and target validation, but also highly valuable as a strategy to treat genetic diseases in which decreasing the levels of a mutant protein would favourably alter the phenotype (9). ASO induced protein knockdown is usually achieved by induction of RNase H endonuclease activity that cleaves the RNA – DNA heteroduplex, leading to the degradation of target mRNA while leaving the ASO intact (20). Another ASO mechanism include translational arrest by steric hindrance of ribosomal activity, interference with mRNA maturation by inhibiting splicing and destabilization of pre-mRNA in the nucleus (21).
Since ASO first use, many chemical modifications have been made to improve resistance to nucleases and consequently, to improve their function and to limit their toxicity. One of the most successful modifications was the substitution of ribose moieties by morpholino moieties. Morpholinos, also known as phosphorodiamidate morpholino oligos (PMOs), normally consist of a 25 nucleotides chain with high specificity, water solubility and resistance to a wide range of nucleases (22,23). Morpholinos do not freely cross the cells’ membrane, their entry in the cell is made by endocitosis and need to be aided by a delivery mechanism, either electroporation, conjugation with a weakly-basic polyamine (ethoxylated polyethylenimine (EPEI)) or the commercially available agent Endo-Porter® (24). Due to this fact, morpholinos cannot be used in in vivo experiments. In order to circunvent this problem, another modification was made through the attachment of a morpholino molecule to a transporter, a dendritic structure called vivo-morpholino that allows an effective cell membrane penetration (25,26).
Locked nucleic acids (LNAs) are another type of oligonucleotides widely used. LNAs present a rigid structure with resemblance with RNA, high afinity with complementary RNA as well as ssDNA, high specificity toward the target, high stability in vivo, lack of toxicity and good resistance to degradation by 3’-exonucleases. In addition, it is possible to synthetize oligomers with lower sizes (6 to 20 nucleotides) and with varied ratios of LNA, allowing different mixtures like LNA/LNA, LNA/DNA and LNA/RNA. LNAs act mainly by recruiting RNase H to degrade the aberrant mRNA, although non-RNase H based mechanisms have also been reported (27).
Currently, the antisense oligonucleotide Vitravene® (ISIS Pharmaceuticals) and Kynamro® (ISIS Pharmaceuticals) have been approved for the treatment of cytomegalovirus infection and homozygous familial hypercholesterolemia, respectively, and other AONs are already in phase 2 and 3 of clinical trials (28). These advances in antisense therapy are encouraging and contributing to the use of ASO in metabolic disorders.
1.3. Pharmacological Chaperones and Proteostasis Regulators
Pharmacological Chaperones (PC) and Proteostasis Regulators (PR) are two classes of distinct small molecules that can correct proteostasis deficiency through the stabilization of nonfuncional or partial functional unstable proteins, originated by specific mutations. Pharmacologic chaperones are small molecules which bind to and stabilize the folded state of a specific misfolding-prone protein, thereby increasing the concentration of the folded mutant protein that can engage its trafficking receptor and proceed to its destination environment, resulting in increased function (29) . It is considered a heterogeneous group of small compounds as it comprises molecules that bind weakly to a specific target protein, competitive inhibitors, ligands, agonists/antagonists and protein cofactors including metal ions. Interestingly, interactions with cofactors, either covalent or non-covalent bonds, are among those that contribute significantly to the maintenance of the terciary structure of a protein (30). The downside of this approach is the small molecules specificity as they have to be tailored for each non-homologous protein linked to a loss-of-function misfolding disease, which number exceed 50 (29). Currently, PC have been developed for several inherited metabolic disorders such as Gaucher disease (31), infantile Batten disease (32) and in Fabry disease (33), which means that they have been adopted as a therapeutic strategy to ameliorate protein misfolding diseases.
When it comes to small molecules to rescue folding defects in proteins, the term chemical chaperone often arises. The slight difference between chemical and pharmacological chaperones comes down to the unspecific action for the former and the more specific direct action, over a particular target protein, observed in the latter (30). Chemical chaperones are mainly osmolytes, protein stabilizers which include polyols (e.g. glycerol, sorbitol), sugars (e.g. trehalose), methylamines (e.g. trimethylamine-N-oxide), free amino acids (glycine, taurin) or its derivatives (e.g. ectoine and gama-aminobutyric acid) and also other low molecular weight compounds with a chaperone-like function, such as dimethyl sulfoxide (DMSO) (30). As mentioned before, chemical chaperones have an unspecific mode of action and do not bind directly to proteins, their action results from the hydration effect which results from the ability of water molecules to establish favourable interactions with polar groups from the protein backbone, thus increasing protein compactness (34,35). Chemical chaperones has shown being effective in rescuing misfolding proteins in diseases such as cystic fibrosis, characterized by the impairment of a transmembranar protein: cystic fibrosis membrane conductance regulator (36), and also in several mild forms of phenylketonuria (37,38).
Proteostasis Regulatores, also called molecular chaperones, are defined as any protein that interacts with and aids in the folding or assembly of another protein without being part of its final structure (39). As central elements of the proteostasis network (PN), which is considered a fully integrated, layered system, unique to each cell type/compartment (40), comprising nearly 1000 molecular chaperones, they regulate protein synthesis, folding, trafficking, disaggregation and degradation (29). The PN is used by cells to respond to proteome insults through stress sensors and inducible pathways which include the heat shock response (HSR), the unfolded protein response (UPR), oxidative stress pathways and growth factor and diet sensitive pathways (41–43). For this reason, PR are designated as stress proteins or heat shock proteins (Hsp) and were initially named according to their molecular weight monomers (Hsp40, Hsp60, Hsp70, Hsp90, Hsp110 and small Hsp), although most of them exist as oligomers. Based on the sort of interaction with client proteins, PR are also classified in holdases, foldases, and disaggregases (44). Foldases and disaggregases are molecular chaperones that function broadly in de novo folding and refolding (i.e. the chaperonins, Hsp70s and Hsp90s), are ATP regulated and recognize segments of exposed hydrophobic amino acid residues, which are later buried in the interior of the natively folded protein. Binding to hydrophobic segments enables these chaperones to recognize the non-native states of many different proteins. Folding is then promoted during ATP- and cochaperone-regulated cycles of binding and release of non-native protein. In this mechanism of kinetic partitioning, (re)binding to chaperones blocks aggregation and reduces the concentration of free folding intermediates, whereas transient release of bound hydrophobic regions is necessary for folding to proceed. Holdades are ATP-independent chaperones (such as the small Hsps) that buffer aggregation, which can recognize and stabilize partially folded proteins, preventing their aggregation and presenting client proteins to foldases (44–46).
Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that unbalanced cell proteostasis are in the origin of pathological states related to a wide range of diseases such as Alzheimer’s, Parkinson’s and metabolic disorders. Hence, while a decline of the proteostasis network is detrimental to cell and organismal health, a controlled perturbation of this network may offer new therapeutic avenues against human diseases (47,48).
(Hsp90; Hsp70 and SQSTM1 – falar mais acerca da sua função? Visto que foram os utilizados na co-IP)
2. Non-Mutation Specific Therapy
The development of strategies where mutation nature is not considered has also showed very interesting outcomes in a wide range of genetic diseases, including metabolic disorders, neurodegenerative, hematological, immunological and ocular. This type of therapeutic approach is considered transversal and effective against different type of genetic diseases, for which the gene causing the underlying condition has been identified, regardless how well the underlying pathophysiology is understood. Hence, non-mutation specific therapies emerge as a promise of providing lasting therapies and even cures for diseases that were previously untreatable.
2.1. Gene Therapy
Gene therapy is the delivery of genetic material into an individual’s cells and tissues to treat inherited or acquired diseases. It is seen as a promising molecular approach that directly delivers into the host cell nucleus a gene aimed to repair the genetic defect, in order to cure or ameliorate the clinical phenotype (49,50). Although, gene therapy can provide treatment for complex genetic diseases and acquired genetic diseases, its ideal target are monogenic diseases (50,51). According to literature, gene therapy has been shown to be effective in treating metabolic diseases (52), immunodeficiencies (53,54), eye (55) and coagulation disorders (56). These type of disorders are characterized by the dysfunction of a single specific gene, where the complete absence or the reduction in activity of the gene product causes the pathological phenotype, thus, the introduction of one or more copies of the healthy gene is able to restore the genetic defect (57).
A number of methods have been stablished to accomplish gene delivery, taking into account not only characteristics of the different genetic diseases, such as the size of therapeutic gene to be transferred and the tissue affected, but also several characteristics of what is an ideal vector: efficient and specific transduction of the target cell regardless of cell cycle, a therapeutic level and proper duration of gene expression, no associated genotoxicity, absent pre-existing immunity against the vector and transgene and a non-invasive delivery route (52).
Effective strategies for clinical gene therapy are based in either in vivo or ex vivo gene delivery methods. The in vivo method involves direct introduction of vector, carrying the therapeutic gene, into the patient, either into or near the target organ. Although this procedure might lead to inadvertent gene transfer into tissues and cell types that are not proper targets and may elicit immune responses towards the transgene and the vector or even damage healthy genes, may be ideal for metabolic diseases for which liver transplantation is a treatment (58). The ex vivo gene therapy implies the isolation of target cells, from donors or patients, to be genetically modified in vitro. Cells are harvested and transduced with a vector to express the therapeutic gene at normal or even supra-normal levels. Subsequently, to restore the healthy phenotype, the gene-corrected cells are infused into the patient where they can proliferate (51). This approach is appropriate for tissue specific diseases for which cell expansion or transplantation have been stablished as a treatment and for this reason has largely been focused on genetic diseases that involved hematopoetic derived cells (59,60).
Different types of gene delivery systems have been exploited as useful vehicles for gene delivery: viral and non-viral vectors. The most common viral vectors are derived from pathogenic viruses: retroviruses, lentiviruses, adenoviruses and adeno-associated viruses (52). Actually, viral vectors arise from its innate ability of deliver genetic material into infected cells. The principles for the generation of viral vectors are the elimination of the viral toxic and infective functions, without altering the capacity to efficiently infect cells and deliver new transgenes (61).
Non-viral vectors, when compared with viral vectors, tend to be less immunogenic, to have larger packing capacity and easier to produce, however the cellular uptake in not very efficient. Several strategies, such as the use of polymeric systems and compounds and physical delivery methods have been used in order to overcome non-viral vectors limitations. Those efforts may some day result in a shift from the use of viral delivery to the use of non-viral delivery, but, in the present, viral delivery the preferred treatment for genetic diseases (52).
In the field of gene therapy new vectors are constantly under development with the aim of targeted integration and DNA editing. At the beginning, early clinical trials for gene therapy were initiated with the hope that a new era for the treatment of inherited diseases would begin, but, only more recently, gene therapy clinical trials have demonstrated safety and efficacy in treating genetic diseases. Gene therapy is certain to represent a new and exciting therapeutic option, when one considers that there are no or inadequate treatment for a majority of patients with genetic diseases (52).
2.2. Enzymatic Replacement Therapy
Enzymatic Replacement Therapy (ERT) is a medical treatment to reintroduce an enzyme into a patient who has deficiency of a specific enzyme, usually associated with inherited metabolic diseases. The concept of systemic delivery of a deficient enzyme, to rescue cellular function, first started with Lysosomal Storage Diseases (LSD) and it derives from early cell-culture experiments by Neufeld and her group (57,62). ERT is usually performed by infusions of an enzyme that is purified from human or animal tissue or blood or produced by novel recombinant techniques. Typically, the enzyme is modified to allow for a longer half-life, more potent activity, resistance to degradation or targeting to a specific organ, tissue or cell type (63). One of the first successful enzyme replacement therapies was for alpha-1-antitrypsine (A1AT) deficiency using derived purified human A1AT (64) and the second one was developed for type I Gaucher disease – an inherited deficiency of lysosomal acid β-glucocerebrosidase that leads to accumulation of the substrate in lysosomes – using, initially, highly purified placenta-derived glucocerebrosidase and later on recombinant technology, constituting now the standard therapy for this disease (65).
Currently, ERT is also available to treat other enzyme deficiency syndromes such as Fabry disease, Pompe disease, Hurler and Hunter syndrome, lysosomal acid lipase deficiency and several of the rarer forms of mucopolysaccharidoses (63).
Despite the advances in ERT, its usefulness is limited due to the fact that a given enzyme preparation does not have beneficial effects on all aspects of a disorder in the same degree, so it is important to remember that this is a treatment with partial effects in specific body compartments (66). For instance, treatment involving modifications of enzymes (e.g. through the addition of mannose-6-phosphate) to target certain tissues (e.g. the heart and kidney) is ill suited to targeting others (e.g. skeletal muscle) that have inherently poor uptake potential owing to low or inexistent expression of the relevant receptor, thus phenotype-genotype correlations also need to be considered (57). Besides that, no enzyme given intravenously crosses the blood-brain barrier, which makes ERT inappropriate for metabolic diseases that affects the brain (67).
Emerging strategies to mitigate ERT limitations include the use of immune tolerance regimens (68), covalent PEG attachment strategies (69), modified targeting procedures or complementary therapeutic methods, such as those involving pharmacologic chaperones or substrate reducing agents (70,71).