Several therapeutic approaches using viruses as delivery platforms are emerging for the treatment of diseases of the central nervous system. Gene therapy bears promise for the treatment of several paediatric neurological conditions, especially those associated with a single gene. Onasemnogene abeparvovec-xioi, a gene therapy product for the treatment of spinal muscular atrophy is already approved for clinical use. Several other gene therapy products are in clinical trials for conditions including, but not limited to, other neuromuscular disorders, lysosomal storage diseases, and leukodystrophies. Many more gene therapy programmes are currently in preclinical development. Viruses as vectors aim to deliver therapeutic protein coding sequences, small RNAs, or tools of genome engineering in the non-dividing cells of the central nervous system. Among those, adeno-associated viruses are becoming increasingly popular vectors, especially for in vivo gene therapy. For ex vivo gene therapy, lentiviruses are more commonly used. During both preclinical and clinical stages, several pitfalls related to the safety or efficacy of such treatments need to be addressed. Improved vectors and gene therapy constructs are being developed and challenges associated with the host’s immune reaction to the vector, transduction capability, and long-term expression of the transgenes are being tackled by scientists and investigators worldwide. A new era for gene therapy lies ahead, which holds the potential to revolutionise clinical care. Overall, there is need for readiness in light of the rising number of clinical trials; validation of outcome measures and sensitive biomarkers will contribute to the successful assessment of the efficacy of gene therapies in clinical testing. Keywords: Adeno-associated virus, adenovirus, cell therapy, central nervous system, gene therapy, lysosomal storage disorders, neuromuscular diseases
The concept of introducing genetic material into a patient’s cells to treat a disease dates back to the 1960s . Over the years, the term ‘gene therapy’ has expanded to include a significant number of therapeutic modalities and approaches; the fruits of breakthroughs in the field of biomedical research. Even though gene therapies were a novel concept only a few decades ago, these innovative approaches have now reached the bedside of patients and are presently being used to treat devastating genetic diseases. Genetic therapies have the potential to revolutionise healthcare practices, cure rare diseases, and make personalised medicine a reality. However, their development holds several pitfalls and challenges.
After Theodore Friedman introduced them in 1972 , the first human trials took place using viruses as delivery platforms to introduce the genetic material into patients’ cells. The results were mostly encouraging, with the exception of two serious adverse events. The first of these was the death of an 18-year-old boy who was treated with gene therapy for ornithine transcarbamylase deficiency, an enzyme deficiency which leads to hyperammonaemia (increased ammonia in the blood). His death was attributed to a virus-triggered exacerbated inflammatory response and multiorgan failure. The second event was the discovery that patients treated for a condition called X-linked severe combined immunodeficiency were developing leukaemia, which was attributed to the insertion of the viral genome into their own genome –.
However, major breakthroughs in the intervening years have dramatically improved the safety and efficacy profile of gene therapies. A new era of clinical development lies ahead. These therapeutic approaches bear promise for the treatment of several devastating paediatric genetic diseases.
There is an unmet need with regards to the treatment of several paediatric genetic diseases. Some of these diseases – which also affect the nervous system – have a known genetic cause that is sometimes associated with only a single gene. Gene therapy has the potential to provide a disease-modifying effect for such conditions in contrast to the symptom-alleviating therapies presently applied to them. Nevertheless, despite recent breakthroughs in biomedical research, gene therapies are not largely available to patients. Applications of gene therapies in human clinical trials have raised concerns about both short-term and long-term safety and revealed several challenges related to application and efficacy. Only a small number of drugs has been approved. Nevertheless, clinical data suggest that gene therapies still hold promise. In this article, I review the viruses being used as vectors in gene therapy and the challenges associated with this type of treatment. I also present key results from the clinical development of gene therapy products – approved or under assessment – for paediatric neurological conditions.
2. Gene therapies: principles and current landscape 2.1 Principles of gene therapies and viral vectors
As per the European Medicines Association definition, the term ‘gene therapy’ refers to the introduction of recombinant genetic material to a patient’s cells to replace, regulate, modify, or add to a genetic sequence, with a view to treat, prevent, or even diagnose a disease . Mechanisms by which gene therapies aim to treat diseases include, but are not restricted to , : (a) Gene replacement, in cases where deficiency or loss of function of a gene product is the cause of the disease; (b) Gene silencing, in cases where the product of a gene is toxic or alters the normal cellular physiology;
(c) Gene editing, in cases where modifications of a specific area of a patient’s genome can lead to a functional gene product and restoration of cellular function; (d) Gene addition, in cases where (over)expression of the transgene product can restore cellular function. The delivery of this therapeutic genetic material to the patient’s cells can be mediated by various vectors (i.e. delivery systems), which can be viral or non-viral (e.g. DNA plasmids, liposomes). This review focuses on the viral vectors used for the treatment of paediatric neurological diseases. Viral-mediated gene therapies can be divided into two main categories: in vivo (Latin: ‘within the living organism’) and ex vivo (‘outside the living organism’). For in vivo gene therapy, the viral vector carrying the transgene (the genetic material of interest) is administered into the patient via the most appropriate route of administration; sometimes, this entails delivering the transgene directly into the tissue of choice e.g. intramuscularly (in the muscle) or intrathecally (directly into the cerebrospinal fluid of the patient) [9, 10]. In ex vivo gene therapy, extracted patient or donor cells undergo programming outside the body by a viral vector carrying the therapeutic transgene. The cells are then re-introduced to the patient via autologous transplantation when the cell source is the patient or allogeneic transplantation if the source is the donor [11, 12]. A viral vector with a gene therapy construct consists of three components: the capsid and/or the envelope of the viral vector (i.e. external part of the virus); the transgene of choice; and the ‘regulatory cassette’ which may include the promoter, the enhancer, or other auxiliary regulatory elements . These components bear distinct characteristics, which can account for both the safety and the efficacy of a gene therapy product. For example, the capsid of a viral vector can determine the cell/tissue specificity of a vector, as well as its immunogenicity – meaning the ability to trigger the host immune response. In contrast, the promoter, together with the other regulatory elements, can create spatial and temporal control over the expression of the transgene. Several types of viruses have been investigated as candidate vectors to deliver gene therapies in the central nervous system . Amongst the most commonly used viruses in the preclinical (animal studies) and clinical (human studies) development of gene therapies are adenoviruses, adeno-associated viruses (AAVs), and lentiviruses, as detailed below: Adenoviruses. Adenoviruses are viruses with a linear double-stranded DNA. Adenoviruses are known to cause human infections, usually of the respiratory system. They have a genome of 26 to 45 Kb depending on the serotype, and they can incorporate large transgenes in their genome. Adenoviruses can transduce both diving and non-diving cells at high transduction rates due to the existence of cell surface receptors that facilitate their internalisation into human cells. Their genome gets stabilised to become extrachromosomal episomes; episomes which do not integrate into the host DNA. This stabilisation is favourable from a safety perspective when compared to the viral vectors whose DNA integrates into the host genome, sometimes in an uncontrolled way, which might affect significant coding areas. The main disadvantage of adenoviruses is their immunogenicity; however, this characteristic favours other applications of adenoviruses, like vaccines and anti-cancer therapies , –. Adeno-associated viruses. AVVs, on the other hand, are not known to cause human disease and they are included among the most promising viral vectors, mostly because of their better safety profile. AAVs are helper-dependent viruses, meaning that they require the presence of helper viruses (e.g. adenoviruses, herpes simplex viruses) to enter a productive infection. In the absence of helper virus, they enter the so-called ‘latent phase’ during which AAVs can also integrate into a genomic locus known as AAVS1 on chromosome 19 in human cells. AAVs have a small, single-stranded DNA which limits their packaging capacity to approximately 4.7 Kb. Unfortunately, this can be a limiting factor for the treatment of many diseases, such as the neuromuscular disease Duchenne muscular dystrophy (DMD) which is caused by mutations in the dystrophin gene, one of the largest of the human genome . They can transduce both dividing and non-dividing cells. The AAV genome is flanked by two T-shaped inverted terminal repeats (ITRs), which act as self-priming areas for the replication. Lab-modified AAVs, also called recombinant AAVs (rAAVs), are usually deficient for all the viral coding sequences except for the ITRs and carry only the coding sequence of interest and the other regulatory elements. By removing the other viral coding elements, the proteins required for integration of the AAV into the host genome are no longer present, adding an advantage to their safety profile. AAVs modified to carry the transgene are recognised by cell surface receptors and get internalised in the cells by a process called endocytosis. They are then trafficked intracellularly in vehicles called endosomes and lysosomes, before entering the nucleus. After uncoating, their genome gets released and undergoes second strand synthesis (the conversion of single-stranded to double-stranded DNA) using the host’s cellular machinery; second strand synthesis is required for gene expression. Self-complementary AAVs (scAAVs) have been engineered to expedite the step of second strand synthesis, which can be rate-limiting for rAAV transduction. The double-stranded genome of rAAVs is usually stabilised as non-replicating extrachromosomal episomes, which are gradually lost with cell divisions. However, at very low frequencies, rAAVs integrate into the host genome , , , . Different rAAVs serotypes have different transduction efficiency or cellular tropism. As shown by studies in animal models, rAAV9 has the greatest transduction efficiency for neurons throughout the brain after both intraparenchymal (directly in the brain) and intravenous administration . Lentiviruses. Lentiviruses are single-stranded RNA retroviruses, which can transduce both dividing and non-dividing cells. A well-known virus of this category is the human immunodeficiency virus (HIV), known to cause acquired immune deficiency syndrome (AIDS). Inside the host cell, the genome of lentiviruses undergoes reverse transcription to produce double-stranded DNA by an enzyme coded from the viral genome, called ‘reverse transcriptase’. This double-stranded DNA can integrate into the host genome facilitated by another enzyme, called ‘viral integrase’. The integrated viral genome replicates, together with the host genome, and this allows long-term (sustained) expression of the transgene. However, the former comes with safety concerns; integration in the host genome can cause insertional mutagenesis of unknown significance depending on the site. This, in combination with the difficulty in producing the high titre required for body-wide application, makes them less preferred vectors for in vivo gene therapy compared to AAVs. Some lentiviruses are non-integrating, e.g. integration-deficient lentivirus vectors, and they have been investigated as vectors of a better safety profile. Nevertheless, lentiviruses are commonly used in ex vivo applications due to their ability to permanently integrate in the host cell genome with low risk of inducing genotoxicity –, .
Adenoviruses, AAVs, and lentiviruses have different profiles of advantages and disadvantages (e.g. safety, transduction ability, immunogenicity ). Overall, in clinical trials of gene therapies for paediatric neurological diseases, AAV vectors prevail. In other fields like oncology and vaccines, adenoviruses are by far the most utilised vectors , .
2.2 Clinical development: the current landscape
Currently, gene and cell therapies using viral vectors are in clinical development for several types of paediatric neurological disorders including neuromuscular diseases, lysosomal storage diseases, leukodystrophies, neurotransmitter diseases, and epilepsy. Overall, genetic disorders associated with a single gene (monogenic disorders) can be a feasible target for gene therapies . This section of the article describes some key clinical developments.
The recent approvement of onasemnogene abeparvovec-xioi (brand name: Zolgensma®) by both the US Food and Drug Administration and the European Medicines Association for the treatment of spinal muscular atrophy (SMA), a rare genetic neuromuscular disease that leads to motor neuron death and muscle atrophy, has raised hope for the treatment of other rare genetic neurological disorders. Onasemnogene abeparvovec-xioi uses a scAAV serotype 9. In the human trial that led to the approval of onasemnogene abeparvovec-xioi, the gene therapy product significantly improved patient survival rate and motor function, and reduced the need for respiratory and nutritional support, especially for the high dosage schemes. However, some safety concerns arose during both the clinical trials and the post-marketing surveillance of adverse events observed in treated patients; these include transient elevation of liver enzymes, transient decrease in platelet number, and thrombotic microangiopathy .
Another example disease is Duchenne muscular dystrophy (DMD), also a rare neuromuscular disease caused by a lack of the protein dystrophin in muscles, which ultimately results in muscle degeneration, loss of ambulation, cardiomyopathy, and premature death in patients. Several isoforms of dystrophins are expressed in the central nervous system, and a large proportion of patients have cognitive involvement . The dystrophin coding gene is one of the largest genes in the human genome, which renders its packaging into a viral vector challenging. However, researchers found an opportunity in Becker muscular dystrophy, another dystrophy caused by mutations of the dystrophin gene, with the difference that a shorter, still partially functional protein is naturally produced. Inspired by this natural variation, they created the so-called micro-/mini-dystrophins, which are smaller in size but still code for the critical parts of the dystrophin.
The first clinical trials of AAV-mediated delivery of the micro-/mini-dystrophins directly into patients’ muscles or intravenously demonstrated increased dystrophin production and improved motor function for DMD patients, but larger trials with a higher number of participants and a longer duration will be required to affirm these findings , . For DMD, another three alternative gene therapy approaches are being developed; these aim to deliver genes of proteins (GALGT2 and follistatin), which can ameliorate DMD pathology if their levels are increased, and sequences for skipping the mutated part of the dystrophin gene and restoring the reading frame (i.e. exon skipping approaches) (review, manuscript under peer-review).
Other neuromuscular disorders for which gene therapies have reached human trials are limb-girdle muscular dystrophy and X-linked myotubular myopathy (XLMTM). In a clinical trial for XLMTM, three patients died from liver failure and sepsis, which raised major concerns with regards to the safety of viral vectors. All three patients were older, and therefore required higher doses .
Lysosomal storage diseases are a group of diseases caused by deficiency of a protein, usually an enzyme, which results in toxic accumulation of metabolic products and progressive disease. This category of diseases includes late infantile neuronal ceroid lipofuscinosis, mucopolysaccharidoses, and gangliosidosis. Therapies for this category of diseases are already in clinical development .
Canavan disease is a leukodystrophy caused by deficiency of the aspartoacylase enzyme required for myelination, which results in accumulation of N-acetylaspartate and leads to spongiform degeneration of the central nervous system. It was one of the first leukodystrophies treated with an AAV serotype 2 (AAV2)-mediated gene replacement with intraparenchymal delivery. Data from the five-year follow up of treated patients suggest a decrease in N-acetylaspartate levels and slower progression or stability of the otherwise observed brain atrophy, with some improvement or stabilisation of clinical features. The biggest effect was observed for patients treated early on, as compared to those with significant brain atrophy at the start of the treatment .
For leukodystrophies, both in vivo and ex vivo gene therapies have been applied. Ex vivo lentiviral programming of haemopoietic stem cells and autologous transplantation has been attempted for both X-linked adrenoleukodystrophy (X-ALD) and metachromatic leukodystrophy (MLD) , .MLD is a fatal demyelinating lysosomal disease, caused by deficiency of an enzyme called arysulfatase A. Based on the data of an interim analysis of a phase I/II trial, patients treated with ex vivo gene therapy for MLD had the needed enzyme expressed in both the haemopoietic stem cells and the cerebrospinal fluid. However, the therapeutic benefit was seen with patients who had not developed symptoms or were at early stages of MLD , indicating again the importance of early intervention, which in the first place required methods for early detection.
Gene therapies are now in clinical development for several other types of diseases including epilepsy or neurotransmitter diseases. For a full review of ongoing clinical trials please refer to Table. Gene therapy is also at the preclinical stages for many other rare neurogenetic diseases, like Angelman syndrome .
3. Challenges of genetic therapies
The ideal viral vector is one that is safe and transduces the target cells with high specificity and at adequate rates via the less invasive route of administration. Additionally, the viral vector should allow sustained expression of the transgene over time with absent or minimal ‘off-target’ effects or other adverse events. Several aspects of viral vectors affect the safety and efficacy of gene therapies . Some of the main challenges of viral-mediated gene therapy are presented below:
Immunogenicity, cytotoxicity, and adverse events. The host immune reaction against the viral vectors, the gene therapy constructs, and the transduced cells is one of the main limiting factors of efficacy and can also raise major safety concerns. Adenoviruses, for which high percentages of immunity exist in the general population, can trigger an intense inflammatory response. In contrast to adenoviruses, AAVs have a better profile with regards to immunogenicity. Ways to tackle immune responses include dose-control, modifications of viral capsids and genome, choice of serotypes known to be less immunogenic, as well as the inclusion of patients with no developed immunity against the used vector. Immunosuppression (reducing the activity of the immune system, e.g. via corticosteroids) has been used for patients in clinical trials and in clinical settings before the administration of the gene therapies .
‘Off-target’ effects.One of the biggest safety concerns associated with the use of viral vectors for genetic therapies is the possibility of the so-called ‘off-target’ events caused by the integration of the viral genome into the host genome. Uncontrolled integration (i.e. insertional mutagenesis) can lead to severe consequences, as critical areas of the human genome can be affected by these events. From this perspective, viral vectors which remain as extrachromosomal episomes (like the adenoviruses and the AAVs) present an advantage. Researchers are looking into the possibility of creating vectors with controlled insertional sites. The long-term off-target effects will need to be carefully considered; therefore, close follow-up of treated patients is of utmost importance .
The route of administration and cell/tissue specificity. The route of administration is very much dependent on the anatomical area of the cells, which require to be transduced. In some neurological diseases, the transgene is required by only a subset of cells in the nervous system (e.g. SMA). For others, transduction throughout the brain is required, as multiple cells in different areas of the central nervous system are affected (as in the case of Angelman syndrome). Some AAV serotypes can cross the blood–brain barrier; therefore, they can be administered intravenously, which is a more appealing route of administration especially in cases where re-dosing is required. However, intravenous administration entails other manufacturing disadvantages, as well as the need for higher doses for adequate levels to be achieved. For central nervous system diseases, direct routes of administration include intraparenchymal (directly into the brain), subarachnoid, or intraventricular (in the cerebrospinal fluid). For the stereotactic intraparenchymal, it is common that vectors remain around the area of the needle. Distinct types and serotypes of viruses have different specificity or affinity for different cells. For example, AAV-9 is known to transduce neurons throughout the brain; therefore, it is a preferred vector for central nervous system diseases . Figure 1 shows the different modes of administration for different diseases:
Figure 1. Route of administration for the different gene therapy products in clinical development. Created with BioRender.com.
Stability of expression over time and the issue of re-dosing. The duration of transgene expression in host cells is one of the determining factors of efficacy. Overall, chromosomal integration of the transgene in the chromosomes (patient’s genetic material) is associated with a more stable long-term expression of the transgene. Non-sustained expression and need of re-dosing with the same vector can be challenging due to the already developed immune response by the host , .
Several viral-based gene therapies for paediatric neurological diseases have reached clinical trials. There is a substantial number of preclinical developments for many paediatric neurological diseases, many of which are rare diseases. The clinical data we have in our hands so far, especially from trials on neuromuscular diseases and leukodystrophies, demonstrate that gene therapies can be an efficacious and promising treatment for many more paediatric neurological conditions. However, human applications of gene therapies have taught researchers several lessons on the characteristics of the vectors and the gene therapy constructs, as well as on the practical and clinical aspects of delivery to patients. Several pitfalls in the preclinical and clinical phases of development of these treatments need to be addressed to ensure their safe application.
Meanwhile, in clinical development, there is an unmet need for the design of more effective clinical trials, especially for rarer diseases. Decisions of efficacy need to be based on sensitive and disease-specific outcome measures, which are both assessor-objective and meaningful for the patients and their families. The same outcomes are those which will facilitate approvals from regulatory authorities, as well as compensations for all patients when a treatment is approved. Additionally, the first clinical data from human trials of genetic therapies for paediatric neurological diseases demonstrate the need for intervention within a critical ‘time-window’, during which interventions have the most impact , , . Towards this direction, the community of researchers should be highly aware of the need to plan the development of newborn screening methods for the early detection of diseases, for which treatments will be available.
Gene therapies hold promise for the future of clinical medicine in the field of Paediatric Neurology. Despite the optimism following the positive results of the first bedside applications, researchers must remain critical of these approaches and united in their efforts to address the existing pitfalls and challenges.
Theodora Markati is an Onassis Foundation Scholar (Scholarship ID: F ZQ 040-1/2020-2021).
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