Gene Therapy Strategies: Gene Silencing PDF

7 Gene Therapy Strategies: Gene Silencing It was already mentioned throughout this book that gene therapy was primarily developed as a strategy targeting recessive monogenic disorders caused by non-functional mutant genes, where in theory the simple addition of a functional (non-mutated) copy of t...

7 Gene Therapy Strategies: Gene Silencing It was already mentioned throughout this book that gene therapy was primarily developed as a strategy targeting recessive monogenic disorders caused by non-functional mutant genes, where in theory the simple addition of a functional (non-mutated) copy of the causative gene would be enough to counteract the disease phenotype and cure the disease. Nevertheless, for more complex diseases, the addition of a normal gene is not enough to revert the disease phenotype. For example, in dominantly inherited disorders, the presence of a single abnormal allele is sufficient to lead to disease manifestation, and thus a gene therapy should instead be able to shut down (or silence) the expression of that abnormal gene. Although this strategy is not so linear and easy, the possibility of silencing the expression of mutant genes using gene therapy approaches became possible with the development of antisense oligonucleotides (ASOs) and with the discovery of the RNA interference (RNAi) pathway. Despite several differences that will be explored further in this chapter, gene therapy strategies based on these two tools have been demonstrated to be efficient in treating dominant genetic diseases. More recently, gene editing tools have also been used to perform gene silencing, including at the DNA level, for example by introducing a premature stop codon or by removing the defective gene; however, this and other gene editing approaches will be discussed in Chap. 8. © 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_7 7.1 Antisense Oligonucleotides In a general way, antisense oligonucleoties (ASOs) are synthetic, unmodified or chemically modified, single-stranded nucleic acid molecules with a size ranging from 8 to 50 nucleotides, that hybridize to their messenger RNA (mRNA) target through Watson and Crick base pairing. ASOs were discovered in 1978 by Stephenson and Zamecnik [1], who observed that a tridecamer oligodeoxynucleotide complementary to Rous sarcoma virus 35S RNA was an efficient inhibitor of the translation of viral proteins and thus of the viral replication cycle. Two decades later, in 1998, the FDA approved the first antisense therapy product to treat cytomegalovirus-induced chorioretinitis, named Vitravene, which was developed by Isis Pharmaceuticals [2]. In fact, this company, now known as Ionis Pharmaceuticals, is the principal booster of ASOs technology, currently having two ASO-based therapies approved in the USA and one in Europe (see Chap. 1). 7.1.1 ASOs Generations The first applications of natural DNA-mimicking, unmodified, ASOs demonstrated that the phosphoribose backbone undergoes rapid degradation by exonucleases and endonucleases, thus limiting their clinical potential. Moreover, these unmodified ASOs also demonstrated a weak affinity for their target. These important limita127 7 128 tions led to the development of several chemical modifications in ASOs, aiming also at increasing their efficacy and bioavailability. The different modifications and improvements made to ASOs led to their classification into three generations (Fig. 7.1). The first generation of ASOs was developed by replacing oxygen by a sulfur atom in the phosphate group to form phosphorothioates (PS) or a methyl group to form methylphosphonates [3]. These modifications facilitated the ASOs production, enhanced stability and increased the ASOs plasma half-life, by improving their resistance to nuclease degradation and reducing binding to serum proteins. One important feature distinguishes the two types of first-generation ASOs; the methyl-modified oligonucleotides do not allow RNAse H-mediated cleavage of the target mRNA, whereas the PS-modified oligonucleotides maintain this feature, allowing their use for RNA silencing purposes [4]. Nevertheless, these modifications also resulted in some disadvantages. For example, methyl-modified oligonucleotides showed reduced solubility and cellular uptake (due to the lack of charge), while the PS-modified oligonucleotides had reduced affinity for the target and displayed some toxic effects. The second generation of ASOs aimed to overcome the main limitations of the previous generation, namely by improving the affinity for the target mRNA, increasing ASOs resistance against nuclease degradation and reducing their immunostimulatory activity [3]. The second generation of ASOs was developed through the introduction of alkyl modifications at the 2’-position of the ribose sugar. The most relevant second generation ASOs examples are the 2’-O-methyl (2’-OME) and 2’-O-methoxyethyl (2’-MOE) ASOs. Despite bringing some improvements and advantages, these modifications prevented the recruitment of RNAse H, not being able to induce mRNA degradation and thus exerting their activity by steric blocking [5]. Finally, the third generation of ASOs includes locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino phosphoroamidates (MF) and was developed through the chemical modification of the furanose ring of the Gene Therapy Strategies: Gene Silencing ASOs, along with changes to phosphate linkages [6]. These modifications enhanced stability, strengthened affinity to the target mRNA and improved their pharmacokinetic profiles. However, similarly to the second generation ASOs, third generation ASOs cannot induce RNAse H degradation. As their backbone has no charge, these ASOs do not have affinity for serum proteins, which reduces the unspecific interactions, but, on the other hand, leads to their rapid elimination from the body. Therefore, third generation ASOs require delivery systems that improve their cellular uptake. From this description of each generation of ASOs, it is clear that there are advantages and limitations to each one of them, and therefore their choice as therapy agents should be weighted carefully (Table 7.1). Moreover, despite the fact that the introduced modifications aimed at improving different features of the ASOs, their application as a therapeutic strategy in preclinical or clinical studies is very dependent on the particular molecular targets, cells, disease and several other important aspects that are involved. 7.1.2 I mportant Considerations for the Use of ASOs in Gene Therapy From the different features of each ASOs generation, several issues emerge that are important to appreciate when considering ASOs use in gene therapy. First, depending on their chemical modification, the functional mechanism of ASOs can lead to target mRNA degradation or to a translational arrest, among others. These different functional mechanisms are important in terms of gene therapy application and outcomes and thus should be taken into consideration. Second, the ASOs pharmacokinetic profile depends on their chemical modifications. For example, PS-ASOs exhibit high affinity for plasma proteins, preventing their elimination from the organism, although the increased unspecific interactions reduce their efficiency. On the contrary, neutrally charged ASOs (e.g., MF) have less tendency to bind plasma proteins, reducing Fig. 7.1 Chemical modifications made to the antisense oligonucleotides (ASOs) backbone that are the basis of the different ASOs generations. These modifications were engineered to improve ASOs efficacy, tolerability profile and bioavailability. In the first generation of modified ASOs, the non-bridging oxygen atom of the ASOs backbone was replaced with a sulfur atom (or a methyl group), improving the resistance to nucleases and the cellular uptake. In the second generation, the changes were introduced in the ribose sugar, improving ASOs safety and efficacy profiles. Finally, the third generation of ASOs included the chemical modification of the furanose ring, along with changes to the phosphate linkages. These modifications resulted in enhanced stability, a stronger affinity for the target mRNA and better pharmacokinetic profiles. PNA, MF and LNA 3rd a ++ +++ Steric hindrance Steric hindrance +++ ++ +++ ++ − ++ Higher nuclease resistance, decreased toxicity, improved uptake, higher affinity for the target mRNA Higher nuclease resistance, higher affinity to the target RNA, decreased unspecific interactions with serum proteins Functional Nuclease Target Cellular mechanism resistance Stability affinity uptake Advantages + + Improved resistance to RNAse H + − nucleases, good cellular activitya uptake for PS-ASOs Cannot recruit RNAse H, difficult cellular uptake (due to neutral backbone), rapid clearance from the body Limitations Immune stimulation at high concentrations, reduced target mRNA affinity for the PS-ASOs, low cellular uptake for methyl-­ modified ASOs Cannot recruit RNAse H 7 Only for phosphorothioate Alkyl introduction 2’-OME and 2’-MOE 2nd Chemical modification of the furanose ring along with modification of phosphate linkages Modifications Sulfur or methyl introduction Generation Examples 1st Phosphorothioate (PS) and methylphosphonates Table 7.1 Main features of the different generations of antisense oligonucleotides. 130 Gene Therapy Strategies: Gene Silencing 7.1 Antisense Oligonucleotides unspecific interactions but increasing their clearance. Systemic delivery of ASOs results in a broad distribution to most tissues, with the exception of the central nervous system (CNS), as they do not cross the blood-­brain barrier. To overcome this important limitation, several studies focused on the delivery of ASOs by intrathecal injection, leading to a rapid and broad distribution into the brain and spinal cord [7]. In fact, the ASOs therapy (Spinraza®) currently approved for spinal muscular atrophy (SMA) is based on intrathecal administration [8]. Third, an important limitation for the success of ASOs in clinical applications is their poor cellular uptake due to their negatively charged nature (at least for some types of ASOs). For this reason, delivery of ASOs has also employed different systems that increase cellular entry efficiency, for example electroporation and microinjection [9]. Finally, intracellular trafficking is another important issue for ASOs. Even with the poor cellular uptake, ASOs can enter the cell through endocytosis and eventually need to escape from the endosomes, which is now recognized as the main limiting step in oligonucleotide therapy. Several strategies can be used to overcome this limitation, such as altering the endosomal barrier or modulating intra-endosomal pH [7]. Despite all these considerations, ASOmediated gene therapy is again in the center of the stage, with several therapies being tested for different conditions and many of them in advanced stages of clinical trials. 7.1.3 Functional Mechanisms ASOs can exert their action through different functional ways, which can be categorized into two main types of mechanisms: an RNAse H-dependent mechanism, that leads to mRNA degradation, and RNAse H-independent mechanisms, that act through nucleic acid occupancy only (Fig. 7.2) [10]. In the first type of mechanism, upon base pairing, the ASOs form an RNA-­DNA hybrid with the target mRNA, becoming a substrate for RNAse H, which then cleaves the mRNA and leaves the ASOs intact. However, to mediate this 131 mechanism, ASOs need to have at least a portion of 2’ unmodified nucleotides and, for this reason, only the first generation of ASOs displays this functionality. On the contrary, the second type of mechanisms is based on the occupancy of target mRNA or even of the DNA. Several effects are possible, such as blocking interaction with RNA binding proteins, thus inhibiting translation, or altering RNA processing through the modulation of splicing. Recently, ASOs that inhibit microRNA (miRNA) function were also developed. These ASOs reduce the silencing activity of miRNAs and consequently increase the levels of the mRNAs they target. 7.1.4  SOs Applications in Gene A Therapy Polyglutamine (PolyQ) Diseases Polyglutamine (polyQ) diseases are a group of nine rare neurodegenerative disorders, each caused by an abnormal expansion of the trinucleotide CAG in the respective causative gene, giving rise to an abnormal polyQ tract in the protein therein encoded (Fig. 7.3). These abnormal proteins tend to aggregate, forming insoluble protein aggregates, which are a key hallmark of polyQ diseases. The toxic nature of the aggregate species remains unclear, since it is not completely understood whether they are the cause or the consequence of the progressive neurodegeneration observed in these diseases [11]. The group includes Huntington’s disease (HD), dentatorubral-­pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and six different spinocerebellar ataxia types (SCA1, 2, 3, 6, 7, and 17). These disorders mostly affect the CNS and, so far, there is no available therapy that is capable of delaying or stopping disease progression [12]. Due to their mostly dominant genetics (SBMA is an exception) and the fact that disease is regarded as resulting from a toxic gain-of-fuction by the expanded proteins, the use of gene silencing strategies is a viable therapy option for polyQ diseases, aiming to reduce the expression of the mutant protein. In line with this idea, several preclinical stud- Fig. 7.2 Functional mechanisms of ASOs. ASOs can exert their action in different functional ways, which can be categorized into two main types of mechanisms: an RNAse H-dependent mechanism, which leads to mRNA degradation, and several RNAse H-independent mechanisms, based on the occupancy of the target nucleic acid. In this latter category, several effects can be produced by the ASOs, including the modulation of the splicing process (by interfering with the activity of small nuclear ribonucleoproteins - snRNPs), the inhibition of translation or blocking of microRNAs. 7.1 Antisense Oligonucleotides ies showed the potential of ASOs in specifically reducing mutant polyQ-expanded protein levels and disease-associated abnormalities [13]. Different generations of ASOs have been tested in animal models, thus exploring different functional mechanisms, such as mRNA degradation or translation inhibition (Table 7.2). Overall, these preclinical studies report results that are promising for future clinical studies. In fact, there is an ongoing clinical trial for HD using ASOs from Ionis Pharmaceuticals. The first results showed a safe profile for the therapy and a dose-dependent reduction of mutant huntingtin (the causative protein) in the cerebrospinal fluid [14]. Spinal Muscular Atrophy (SMA) Spinal muscular atrophy (SMA) comprises a group of genetic disorders characterized by degeneration of anterior horn cells and resultant muscle atrophy and weakness. Around 95% of the SMA cases are caused by an autosomal recessive deletion or mutation in the survival of motor neuron (SMN1) gene [15]. SMN2 is another gene form present in humans, responsible for around 10% of the physiological levels of the SMN protein (see Chap. 6 and Fig. 6.2). In SMA patients with the mutation in the SMN1 gene, protein production relies on the SMN2 gene alone, which, despite being low, ensures enough SMN protein for survival. Thus, it is not surprising that the clinical severity of SMA inversely correlates with the SMN2 gene copy number. The presence and features of the SMN2 gene are the basis for the approved ASOs therapy for SMA patients. Spinraza® is a intrathecally delivered 2-MOE-PS oligonucleotide from Ionis Pharmaceuticals, whose functional mechanism is based on modulation of SMN2 gene splicing [8]. Studies using this therapy yielded encouraging results, as the ASOs administration showed a good safety profile and tolerability, leading to its approval by the FDA and EMA. Duchenne Muscular Dystrophy (DMD) Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by progressive muscular degeneration and weakness. It is caused by different mutations (deletions, insertions and point mutations) in the DMD gene, 133 resulting in a prematurely truncated, unstable dystrophin protein [16]. The protein is expressed at the muscle sarcolemma, in a protein complex that links the cytoskeleton to the basal lamina. However, the exact mechanism by which dystrophin deficiency leads to muscle fiber degeneration is not yet completely elucidated [17]. Although DMD is a recessive disease, the large size of the DMD gene, with more than 70 exons (more than 220 kb), makes it unsuitable, or at least challenging, to use a therapeutic strategy that would supply a normal copy of the gene. Interestingly, the functional versatility of ASOs, namely the possibility of modulating splicing, provides an important opportunity for DMD therapy, through the generation of a functional protein by skipping DMD-causing mutations. Based on this versatility, Sarepta Therapeutics developed an ASO-based therapy for DMD, which was approved by the FDA in 2016 under the name Exondys 51™ (eteplirsen). The ASOs in question is a phosphorodiamidate morpholino oligomers that promote specific skipping of DMD exon 51 (in defective gene variants) leading to the restoration of the normal DMD reading frame and the production of a functional dystrophin protein (Fig. 7.4) [18]. A pooled analysis of different studies using this ASOs-based drug revealed an increase in dystrophin-positive fibers in muscles and an improvement of walking distance in treated patients, although the clinical significance of these results is still unclear [19]. Other Diseases The versatility of ASOs functional mechanisms, as well as their easy design and production, makes them attractive therapeutic agents for several diseases. Recently, an ASOs-based product named Tegsedi™ (inotersen) was approved in the USA as a treatment for hereditary transthyretin-­ mediated amyloidosis [20]. Apart from the approved products based on ASOs technology that were described in the above sections, several clinical trials are also currently ongoing for other diseases, including amyotrophic lateral sclerosis, Alzheimer’s disease and frontotemporal dementia. These and other clinical trials highlight the interest and potential of ASOs technology for gene therapy, and new 7 134 Gene Therapy Strategies: Gene Silencing Fig. 7.3 Genes and proteins involved in polyglutamine diseases, highlighting the number of glutamine repetitions associated with the diseases phenotypes. Table 7.2 Studies using antisense oligonucleotides to treat polyglutamine diseases. Disease Huntington’s disease Target mutHTT Chemistry cEt gapmer mutHTT PS-2’OME gapmer mutHTT PS-2’OME cET gapmer mutHTT 2’MOE gapmer mutHTT cET mutHTT PMO HTT PS-2’OME SCA2 Ataxin-2 2’MOE gapmer SCA3/MJD Ataxin-3 PS-2’OME SBMA Androgen receptor cEt gapmer Androgen receptor cEt/2’MOE gapmer HTT Huntingtin ICV intracerebroventricular Mechanism RNAse H-mediated degradation RNAse H-mediated degradation RNAse H-mediated degradation RNAse H-mediated degradation RNAse H-mediated degradation Translation blockade Exon skipping Administration route Reference Intraparenchymal Carroll et al. (2011) RNAse H-mediated degradation Exon skipping ICV RNAse H-mediated degradation RNAse H-mediated degradation Subcutaneous ICV Kordasiewicz et al. (2012) ICV Ostergaard et al. (2013) ICV Stanek et al. (2013) ICV Southwell et al. (2014) ICV Sun et al. (2014) Intraparenchyma Evers et al. (2014) Pulst (2016) ICV ICV Toonen et al. (2017) Lieberman et al. (2014) Sahashi et al. (2015)