Advanced therapies in pediatric genetic diseases

The transfer of a normal copy of a gene to replace the function of a pathological variant is commonly known as gene therapy, the most widely used name to refer to this process. By extension, gene editing and the modulation of a gene through genetic mechanisms are also generally referred to as gene therapy. We ought to highlight that, conceptually, gene transfer refers to the incorporation of a normal version of the gene without acting on the mutated gene. Since DNA is very inefficient at reaching cells and passing into the nucleus, the most efficient way to transfer a gene is through a vector. There are chemical vectors, most commonly cationic lipids that interact with the negatively charged DNA molecule, thus forming a complex to deliver the genetic content to cells. They may carry large molecules of DNA and they exhibit a low immunogenicity. There is increasing investigation of these vectors to increase their efficiency and versatility and to achieve lasting expression in human recipients. There are also viral vectors, which are very efficient in delivering DNA to cells and achieving long-term expression, although they may be highly immunogenic and can cause inflammatory reactions in certain organs.

Usually, viral vectors containing genetic material have three key components: a promoter, a transgene (the genetic material the vector delivers) and a termination signal. It may also contain inverted terminal repeats (ITRs), which serve as the origin of replication for the synthesis of the nucleic acid copy. Viral vectors are natural viruses whose genome has been modified to achieve the desired function. The newly introduced gene can replicate autonomously and is controlled based on how the vector was constructed. The elimination of certain viral components from the vector alters its infectivity. In other words, the vector does not replicate the same way nor triggers the same immune response as the wild-type virus. There are five main types of viral vectors: retroviral and lentiviral vectors (which can carry transgenes of up to 8 kb), herpes simplex virus-based vectors (HSV, containing 40–150 kb of genetic material), adenoviral vectors (8–30 kb) and adeno-associated virus vectors (AAV, up to 5 kb of genetic material). The first two, retroviral and lentiviral vectors, integrate into the genome, which can lead to disruption of the host genetic material and cause harm to the patient. In contrast, HSV-1, adenoviral and adeno-associated virus vectors do not integrate into the host genome and persist in the cell nucleus as extrachromosomal episomes (Fig. 2).11

Figure 2.

Illustration of the mechanism of action of gene therapy for the defective SMN1 gene using AAV9. The vector is taken up by the cell via an endosome, which then breaks down when it reaches the cell nucleus and releases its contents. Note that the vector contains the cDNA of the gene, that is, only the exons, without the introns.

At present, AAV vectors are the leading platform for gene transfer in the treatment of several human diseases. Many aspects have been improved in this approach, increasing its popularity and contributing substantially to the growth of the experimental and clinical field of gene therapy. These episomes replicate autonomously within the nucleus, although independently of the host genome. Administered intravenously, they are distributed to all cells throughout the body and generally can cross the blood-brain barrier (Fig. 2).

The vector enters the cell via endosome, the endosome breaks down in the cytoplasm, and the vector reaches the nucleus, where it delivers the transgene, which forms episomes. If the cell replicates, the episomes (usually 3–6 per cell) are distributed as they would in a cell division process. Thus, the effect is diluted over time until, after a few successive divisions, some of the cells will not receive the episome. In contrast, when delivered to cells that do not replicate, such as neurons, the episomes remain for a prolonged period, although the current data is insufficient to verify their long-term functionality. The viral vector dose varies, but is calculated based on patient weight with limits based on whether the patient is older or an adult. Thus, the dosage is calculated in vector genomes per kilogram of body weight for systemic intravenous administration, for instance, doses of up to 3 × 1014 viral genomes per kg of body weight have been administered for treatment of spinal muscular atrophy.

In respect of the host’s immune response to vector administration, there are important aspects to consider, such as the dose that can generate an initial response in the host and whether the host carries pre-existing neutralizing antibodies resulting from previous infections or, in the case of infants, transferred from the mother (passive immunity). All these factors can affect the effectiveness of the treatment. At present, gene therapy with AAV9-SMN1 (Zolgensma) is authorized by the Food Drug Administration (FDA) and the EMA in patients aged less than 2 years with spinal muscular atrophy, and trials are underway to investigate its intrathecal administration in older patients.12 Several clinical trials are also underway to investigate AAV vectors for management of other neuromuscular and metabolic disorders, among other diseases, which are registered and can be consulted in the ClinicalTrials.gov database.

Gene therapy carries risks, and these need to be taken into account in the selection of the optimal treatment option for the patient. Some of the factors to consider are the severity of disease, the availability of other therapies and the age of the patient. Deaths resulting from serious adverse events of gene therapy have been reported, chiefly in relation to the vector dose, hepatotoxicity and angiopathy. Thus, several trials may be discontinued if such complications occur. In any case, it is important to be well informed and not to create unrealistic expectations when deciding on this type of treatment for patients with serious genetic diseases.13

Gene editing is based on the ability to make very specific changes to the DNA sequence of a living organism and can be used to modify an altered sequence. It is performed using enzymes, particularly nucleases, designed to bind to a specific DNA sequence and capable of cleaving the DNA strands to allow recombination between the existing DNA and the replacement DNA, resulting in the insertion in the genome of the delivered DNA. The key to this gene editing technology is a molecular tool known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats and caspase 9).14

Unlike gene transfer, which entails delivery of a complete gene with normal function, in gene editing it is the host’s altered DNA that is modified to restore wild-type expression or remove the aberrant expression.

Precision gene editing is being investigated in clinical trials for cancer immunotherapy and treatment of viral infections and hereditary hematologic, metabolic and eye disorders. At present, these are very specific clinical applications that achieved a localized effect on somatic cells and not a generalized distribution throughout the body. Thus, it can be used in skeletal muscle, whose cells are postmitotic and multinucleated, which would allow repair of a subpopulation of nuclei potentially beneficial to the entire muscle fiber. It can also target the retina and the liver. Some of the main advantages of gene editing are its versatility, as it can target any specific sequence or cell type, that it can be used to edit single point polymorphisms, deletions, or duplications, and that it could potentially be applied to nearly all genetic diseases. Its main drawbacks, are that its efficiency may be low, that it may modify germ cells so that the edited sequence is passed on to future generations, that it may result in unintended off-target editing of genomic regions other than the desired one, or, in the case of on-target editing, generate genetic mosaicism, and, above all, that its long-term effects are currently unknown. However, there are more than 80 active clinical trials using CRISPR technology, and almost all applications in current clinical trials correspond to localized diseases or target specific organs. CASGEVY, the first gene editing therapy for sickle cell disease and beta thalassemia, has recently been approved at a cost of approximately €2 million per patient. It is an ex vivo therapy that involves editing of blood cells extracted from the patient that are later reinjected. In mitochondrial diseases with heteroplasmy, in which both the variant and the wild-type sequences are present in the same cell, the disease may manifest depending on the number of abnormal copies. In this case, mitochondrial DNA can be modified by delivering nucleases that specifically cleave and eliminate mutant mitochondrial DNA, an effect that has been observed both in heteroplasmic cells and animal models.

Base editors that make base changes via precise C>T or A>G transitions through chemical modification of the bases are currently being used in gene editing experiments and trials. There is also a third generation of caspases that, controlled through an RNA guide, can cleave a single strand (instead of two strands, like the first version of CRISP-Cas9).16 All of these innovations aim at reducing the risks and disadvantages and increase the efficacy of gene editing.

As in the case of gene therapy for spinal muscular atrophy, which involves systemic administration via intravenous injection, the extremely high price tag of all these therapies raises important questions about the feasibility of making them available to the entire patient population and questionable issues in relation to pricing schemes and the financial sustainability of health care systems.15Table 2 presents some of the high-cost therapies currently authorized for use in humans.

Epigenetics refers to anything that changes the expression of a gene without changing its sequence. For example, DNA methylation (addition of methyl groups to the DNA) and histone deacetylation (histones are proteins that interact with chromatin) impede access to the replication machinery so it cannot have access to the template to express the gene. These mechanisms can be modified through pharmacological agents, such as histone deacetylase (HDAC) inhibitors, widely used in cancer research and trials, although their applications in other diseases is still limited. One of the best-known HDAC inhibitors is valproic acid, an anticonvulsant that can also act as an epigenetic modifier on hundreds of genes and that has been used in several clinical trials in the field of spinal muscular atrophy due to its in vitro and in vivo capacity to increase expression of the SMN gene and the production of full-length SMN RNA transcripts in patients with this disease. The results of these trials were disappointing.17 However, it may be a useful addition to the therapeutic armamentarium as a coadjuvant for other disease-modifying therapies.

The function of genes is to carry information so that the body’s proteins can be synthesized through messenger RNA (mRNA). This protein synthesis process takes place in two stages: transcription, which consists of the synthesis of pre-mRNA from DNA, splicing of pre-mRNA and formation of mature mRNA; and translation, which consists of the synthesis of proteins by ribosomes using mRNA as a template.

Pre-mRNA includes exons and introns and undergoes splicing to remove the introns and join the exons together to form mature mRNA. It is synthesized using the antisense strand of DNA as a template. As a reminder, DNA is double-stranded with both strands running antiparallel: each strand has a 5′ end and a 3′ end, with the sense and antisense strands oriented in opposite directions. The single strand of pre-mRNA uses the antisense template to generate a 5′ to 3′ single-stranded sequence with the same directionality as the sense strand. The mature mRNA exits the nucleus and enters the cytoplasm, where ribosomes synthesize the corresponding polypeptide.

Antisense oligonucleotides hybridize with complementary sequences in single-stranded pre-mRNA. These oligonucleotides are short single-stranded molecules of 8–50 bases designed according to the target sequence. Upon binding to pre-mRNA, they modulate splicing by forming a double-stranded segment at the target site.

Although they do not cross the blood-brain barrier, antisense oligonucleotides achieve widespread distribution in the central nervous system (CNS) after intrathecal injection, making them very valuable as therapeutic tools in neuronal diseases.18

It is important to note that if the sequence targets the exon, the exon will be excluded from the mature RNA (exon skipping), as occurs with the dystrophin gene in Duchenne muscular dystrophy therapy. In contrast, hybridization with intronic regions that inhibit inclusion helps with exon inclusion. This has given rise to terms such as exonic splicing enhancer (ESE) or exonic splicing silencer (ESS) as well as intronic splicing enhancer (ISE) or intronic splicing silencer (ISS). This is the mechanism of action of the nusinersen oligonucleotide (Spinraza), of 18 bases, which specifically binds intronic splicing silencer N1 (ISS-N1) in intron 7 of the SMN2 gene. This inhibits binding of two splicing repressors in exon 7 (hnRNPA1 and A2), enabling the inclusion of this exon in the mature RNA. The drug was approved by the FDA in 2016 and by the EMA a year later for treatment of all forms of spinal muscular atrophy.19

Another treatment approach is to reverse or block the toxic effect of a specific RNA that is harmful to the cell. For example, this is being investigated in myotonic dystrophy caused by CTG triplet expansion (CUG in RNA) in the DMPK gene. Following transcription, the expanded CUG repeat in the DMPK mRNA sequester the MBNL1 splicing factor resulting in loss of function. The CUG repeat expansion also leads to a relative gain of function in another splicing factor, CUGBP1. The imbalance between MBNL1 and CUGBP1 results in aberrant splicing events that cause the characteristic symptoms of the disease. The expansions can be blocked with chemical agents, such as pentamidine, which bind the CUG repeats in competition with the splicing factors, thus neutralizing the effect of the repeats and restoring normal splicing.20

It is also possible to block or degrade RNA by means of RNA interference (RNAi). In this case, the intervention works on mature RNA, harnessing an existing biological process by which RNA molecules suppress gene expression of double-stranded RNA genes to regulate mRNA expression through complementary base pairing. An enzyme (Dicer) is used to cleave long double-stranded RNA molecules into several small interfering RNA (siRNA) molecules. One of the siRNA strands is loaded into a protein complex known as RISC (RNA-induced silencing complex) that identifies and cuts the aberrant mRNA, which is then degraded by the cell’s RNA decay machinery. This blocks expression of the aberrant gene. When it comes to its application for human use, a drug based on AAV-RNAi for treatment of Huntington disease is currently undergoing Phase I/II trials (NCT04120493). It uses an AAV5 to deliver a microRNA that targets the huntingtin protein mRNA. These studies will probably yield important safety and feasibility data that may also be relevant for the use of this approach in the treatment of other diseases involving triplet expansion or RNA toxicity

Replacement therapies, whether they replace the gene or its products, may seem like the simplest and most obvious solutions at first glance. However, the replacement mechanisms are not straightforward, as many factors must be taken into account, including molecular weight, route of administration, tissue penetration, half-life, and the metabolism of externally administered proteins. Proteins are usually synthesized artificially (recombinant proteins) given the dangers of pathogen transmission when they are obtained from other sources. Some proteins are difficult to deliver to the target organ or cell, and systemic administration of proteins may also pose problems if they have a large molecular weight.

There are three key steps in the translation of RNA to protein by ribosomes: initiation, elongation and termination. Many neuromuscular diseases involve nonsense mutations in which an amino acid codon is replaced by a stop codon. These premature termination codons prevent the synthesis of the full-length product, resulting in a truncated protein that undergoes rapid degradation. In many cases, the mRNA carrying the stop codon is even degraded before being translated. It is possible to inhibit the reading of premature termination codons with compounds such as aminoglycosides, PTC124, and, more recently, Elox. These drugs have been tested to modify the effect of nonsense mutations through the addition of another amino acid at the site of the termination codon. The transfer RNA carrying the amino acid may not introduce the specific amino acid present in the normal protein, and in many instances transforms a truncated protein (resulting from the nonsense mutation) into a full-length protein that has an amino acid change at that point in the sequence, as if it were a, but this protein will have an amino acid change (as if the defect were a missense mutation). Thus, it has been observed that the use of PTC124 tends to result in the insertion of specific amino acids to replace the stop codon: Gln, Lys or Tyr if the stop codon is UAA or UAG, and Trp, Arg or Cys if the stop codon is UGA.21

Once the protein is synthesized (if the disease mechanism allows the synthesis of an aberrant protein different from the normal one), post-translational modification and function may include transport of the protein to the membrane, forming a complex with other proteins, acting as a catalyst for a reaction, or functioning within a specific metabolic pathway. To perform some of these functions and increase or reduce its stability, the protein may have folds and signals, and its potential functions can be expanded by attaching additional functional groups, such as acetates, sugars (glycosylation) for tagging, recognition or phosphorylation, and disulfide bridges to change its shape. Finally, the protein is metabolized and degraded by different mechanisms through the lysosome, ubiquitins, or proteasome. In this regard, there are medications that target the chemical modifications or degradation of proteins of interest, making this a very promising field of study.22 As mentioned above, ClinicalTrials.gov is a database of publicly and privately funded clinical trials conducted worldwide, where it is possible to consult all medications of this type that are being investigated in humans.