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6 Gene Therapy Strategies: Gene Augmentation As it was already mentioned in the first chapter of this book, but will also be made clear throughout the remaining chapters, the term gene therapy encapsulates different approaches and applications based on the use and delivery of different nucleic aci...

6 Gene Therapy Strategies: Gene Augmentation As it was already mentioned in the first chapter of this book, but will also be made clear throughout the remaining chapters, the term gene therapy encapsulates different approaches and applications based on the use and delivery of different nucleic acids. The most straightforward and perhaps most simple strategy for a gene-based therapy consists in adding a new protein-coding gene, which is an approach called gene augmentation therapy (Fig. 6.1). For monogenic recessive disorders in which the causative mutated is nonfunctional, the therapeutic gene to be delivered will be the normal wild-type form of the gene. The delivery of a correct copy of the gene is expected to restore the production of the defective or missing protein and thus revert the disease phenotype. This type of gene augmentation therapy is often also referred to as gene replacement therapy. As already discussed, this rationale was behind the first gene therapy clinical trial performed and is also at the basis of the majority of the gene therapy clinical trials performed so far. However, for monogenic dominant diseases or more complex multigenic diseases, this approach would not be effective. For these diseases, the use of gene therapy strategies based on gene silencing or editing would be more suitable (these will be discussed in more detail in Chaps. 7 and 8). Nevertheless, gene augmentation strategies can still be used in these more complex diseases, namely by using protein-coding genes that improve cellular function and homeostasis, con- tributing to an improvement of the disease phenotype, although in theory it will not cure the disease or solve its molecular cause. For example, genes codifying for growth factors, cytokines or autophagy-activating proteins have been proven successful as therapeutic agents in several complex diseases. This type of gene therapy is often generally referred to as gene addition therapy. Two important considerations must be made about gene augmentation therapy strategies. First, it is important to take into account the size of the gene to be added, as this will conditionate the choice of vector and therefore the efficiency and safety of the therapy. According to the last annotation of the human genome, on average, a human gene has ~29 kb, which makes packaging entire genes unfeasable for most of the viral vectors commonly used in gene therapy. One form to circumvent this problem is to use only the cDNA, which on average has ~2.5 kb and therefore can be packaged in any viral vector. However, for larger genes, packaging the cDNA in viral vectors will still be virtually impossible. For example, Duchenne muscular dystrophy is caused by mutations in the DMD gene, which codifies the dystrophin protein. The gene has more than 220 kb and its cDNA around 14 kb, which makes it unsuitable, for example, to be packaged into an AAV or a lentiviral vector. A second important consideration to be made is related with the expression levels of the therapeutic gene. This question was © Springer Nature Switzerland AG 2020 C. Nóbrega et al., A Handbook of Gene and Cell Therapy, https://doi.org/10.1007/978-3-030-41333-0_6 117 6 118 Gene Therapy Strategies: Gene Augmentation Fig. 6.1 Gene augmentation therapy, which refers to the introduction of an additional functional gene, aims at compensating a pathological mutation or improving the homeostasis of a compromised cell. already discussed in the first chapter as one of the important aspects that need to be adressed in gene therapy applications. Nevertheless, this question is particularly sensitive in gene augmentation therapy strategies, as the aim is to produce correct/therapeutic levels of protein expression. The following sections develop important ideas and concepts related to gene replacement and gene addition therapy, providing examples of both strategies. 6.1 Gene Replacement Gene replacement therapy can be applied both ex vivo and in vivo. In fact, several currently approved gene therapy products use gene replacement therapy, such as Strimvelis® (ex vivo) and Luxturna® (in vivo). Additionally, there is an extensive list of studies using gene replacement therapy for different diseases both in preclinical and clinical settings. Therefore, it is probable that other gene replacement therapies will be approved in the near future [1]. The single-dose gene replacement therapy (AVXS-101) for spinal muscular atrophy (SMA), based on intravenous delivery by AAV9 and with spectacular results, is an example of a therapy that reached the market very recently [2]. SMA constitutes a group of genetic disorders characterized by the degeneration of anterior horn cells and subsequent muscle atrophy and weakness. There is a wide range of clinical manifestations, different degrees of severity and variances in life expectancy, which led to the classification of different clinical types of SMA (Table 6.1) [3]. The incidence of SMA is of 1 in 10,000 live births, whereas the prevalence of the carrier state is of 1 in 54 [4]. Around 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene [5]. In healthy human subjects there are two forms of the SMN gene: SMN1 and SMN2 (Fig. 6.2). Both genes encode for the SMN protein, but the survival motor neuron 2 (SMN2) gene bears a nucleotide substitution in an exon splicing enhancer that results in the exclusion of exon 7 during transcription. Thus, the protein formed is truncated and nonfunctional, being rapidly degraded. However, the exon 7 exclusion is not complete, and around 10% of the SMN protein encoded by the SMN2 gene is functional. In SMA patients, as the SMN1 6.1 Gene Replacement 119 gene is not f­ unctional, the production of the SMN protein relies only on the SMN2 gene. Consequently, it is not surprising that SMA severity is inversely correlated with the number of SMN2 gene copies [6]. A phase I clinical study revealed the safety and efficacy of an in vivo gene therapy based on the delivery of a functional copy of the SMN1 gene using AAV9 vectors [2]. Table 6.1 Clinical types of spinal muscular atrophy (SMA) and their main features Type 0 1 2 3a 4 Age of onset Before birth 0–6 months 6–18 months 18 months–3 years 10–31 years Life expectancy <1 month <2 years >2 years Adult Adult Maximal motor milestone None None Sitting Walking Normal Number of SMN2 copies 1 2 3–4 3–4 4–8 There is an additional categorization in SMA type 3a, 3b, and 3c, each having a different age of onset a Fig. 6.2 Overview of the molecular mechanism underlying spinal muscular atrophy (SMA). In healthy individuals, the SMN1 gene (survival motor neuron 1) encodes for a functional protein that is essential for motor neuron development. A second gene, SMN2 (survival motor neuron 2), is also present, although it only encodes for a small percentage of the functional protein. In SMA patients, mutations in the SMN1 gene produce a nonfunctional protein; therefore, the only functional protein is produced by the SMN2 gene. That is why the number of copies of the SMN2 gene modifies the severity of the condition. 6 120 The 15 children enrolled in the 2 cohorts of the study were homozygous for the deletion of exon 7 in the SMN1 gene and had 2 copies of the SMN2 gene. A 20-month follow-­up after the gene therapy administration showed that all the children were alive, compared with an 8% survival rate of the natural history cohort. Importantly, no major adverse effects of the therapy were described. From the 12 children that received the higher dose (2.0×1014 vg per kilogram), 11 sat unassisted, 11 were able to feed orally and could speak, and 2 walked independently. These spectacular results and a wide range of preclinical studies opened an important avenue for gene replacement therapies that are in the R&D pipeline. It also led to a historical acquisition, with pharmaceutical company Novartis paying 8.7 billion US dollars for AveXis Inc., which was the company that developed the above phase I study. Recently, in May 2019, the FDA approved this therapy under the name Zolgensma, promising a onetime cure for SMA and with a marketing price of 2.125 million dollars. 6.2 Gene Addition Gene replacement strategies are not suitable for monogenic dominant diseases, as even with an additional copy of the normal gene, the disease phenotype will be maintained. Also, for more complex diseases resulting from the combination of multiple genes and environmental factors, this strategy may have none, or very limited, therapeutic effects. Nevertheless, gene therapy can still represent an important therapeutic option for these diseases, as it may be used to produce an improvement in cellular function and homeostasis, therefore contributing to mitigate the disease phenotype. In fact, several studies both at the preclinical and clinical levels have focused on using gene addition therapy as a therapeutic approach for several complex conditions affecting human health. Different protein-coding genes can be used in these strategies, aiming to modulate diverse cell mechanisms and pathways. This type of strategy is particularly useful in cancer applications, using, for example, genes that arrest cell proliferation, like cyclin-depen- Gene Therapy Strategies: Gene Augmentation dent kinase inhibitors or cell cycle checkpoint regulators, such as p53. In fact, the first gene therapy product ever approved (in 2003) is based on the delivery of p53 as a treatment for different types of cancer [7]. However, Gendicine® (developed by Sibiono GeneTech company) was only approved in China, and its beneficial effects are not completely consensual among the scientific community. Gendicine® was approved for treating head and neck cancer and is based on the expression of wild-type p53, upon direct intratumor, intracavity or intravascular delivery by an adenoviral vector. By 2018, more than 30,000 patients were treated with Gendicine®, and more than 30 clinical trials were published [8]. It was used alone or in combination with chemotherapy and/or radiotherapy for at least five different types of cancer, including nasopharyngeal cancer and hepatocellular carcinoma. Most of the published studies reported no major adverse effects. Despite the number of studies and subjects treated, currently Gendicine® continues to be commercialized exclusively in China. Gene addition-­based delivery of protein-coding genes was also studied as a possible means to modulate other cellular functions and achieve a therapeutic effect, for example aiming to, (i) potentiate the activity of cellular degradation systems (ubiquitin-­proteasome system - UPS) - and autophagy), (ii) increase cellular proteostasis, and (iii) increase the expression of growth factors and cytokines. The use of genes activating autophagy or leading to the expression of chaperones is a gene addition strategy particularly interesting in context of neurodegenerative diseases, where frequently there is an abnormal accumulation of aggregate-prone proteins. Strategies based on the expression of growth factors and cytokines have different goals and target other molecular mechanisms, aiming a protective role by potentiating cell survival or contributing to cellular homeostasis improvement. This idea was the basis for the development of CERE-110, a gene therapy product for Alzheimer’s disease (AD) that relies on the delivery of nerve growth factor (NGF). AD is a progressive neurodegenerative disease characterized by a decline of cognitive functions and memory defects. A neuropathological hallmark of AD is