Gene Therapy Strategies: Gene Augmentation PDF

6.3 Gene Addition to Modulate Autophagy the presence of extracellular amyloid plaques and the intracellular accumulation of hyperphosphorylated Tau protein. A small percentage (~5%) of AD cases display a Mendelian inheritance pattern, although most of the cases are sporadic and associated with late...

6.3 Gene Addition to Modulate Autophagy the presence of extracellular amyloid plaques and the intracellular accumulation of hyperphosphorylated Tau protein. A small percentage (~5%) of AD cases display a Mendelian inheritance pattern, although most of the cases are sporadic and associated with late disease onset (after 65 years) [9, 10]. Despite the huge research efforts towards a therapy for AD, most of the results of the clinical trials have been disappointing, and even the approved drugs (such as memantine or rivastigmine) have very limited efficacy in stopping or reverting the disease progression. Considering the unmet and urgent need for therapies for AD, several studies focused on gene therapy as a possible route. One of the ideas behind gene therapy studies was to increase neuronal protection, trying to prevent or delay the neuronal loss observed in AD. The first strategies focused on the delivery of brain-derived neurotrophic factor (BDNF). A study in rodent and nonhuman primate models of AD showed a protective effect of BNDF expression, preventing neuronal loss and leading to cognitive improvement [11]. Later, the studies’ attention moved to NGF, as a way to prevent the degeneration of cholinergic neurons, which is an early event and an important contributor to AD cognitive decline. A very early study using aged nonhuman primates showed a robust sprouting of cholinergic fibers upon NGF expression [12]. The animals received an intraventricular implant of polymer-encapsulated fibroblasts genetically modified to express human NGF (ex vivo gene therapy). Later, a phase I clinical trial used a similar procedure, transplanting autologous fibroblasts expressing NGF (transduced with a Moloney leukemia viral vector) into the basal forebrain of eight individuals with mild AD. The results showed an improvement in the rate of cognitive decline [13]. In a posterior phase I clinical trial, ten patients received an AAV2-NGF (CERE-110) dose directly injected through stereotaxic surgery into the nucleus basalis of Meynert. A 2-year follow-up demonstrated the safety profile of CERE-110 and showed a reduced rate of cognitive decline [14]. These encouraging results led to the development of a phase II multicenter clinical trial of CERE-110, following the acquisition of Ceregene (that initially developed 121 the product) by Sangamo Therapeutics in 2013. The new clinical trial enrolled 49 patients with mild to moderate AD, from which 26 received a bilateral injection of AAV2-NGF and 23 a sham procedure. The first results of the study were somehow mixed, and 2 years after the procedure both groups showed a similar decline in cognitive function. In 2015, Sangamo Therapeutics ended the development of CERE-110. Despite the failure in CERE-110, the development of gene therapy products based on neurotrophic factors continues. For example, NurOwn, a cell therapy product, uses mesenchymal stem cells (MSCs) expressing NGF as a therapy for amyotrophic lateral sclerosis (ALS), and 3 clinical trials were already completed enrolling 74 patients. The first results showed no adverse effects and indicated mild improvement in the disease symptoms (assessed by specific scales) [15]. A future phase III clinical trial is planned. Other gene addition therapy approaches aim at inducing cellular apoptosis, by delivering genes that trigger this event. This type of strategy, which is commonly referred to as suicide gene therapy, is specifically used for cancer applications and is discussed in further detail in Chap. 9. 6.3  ene Addition to Modulate G Autophagy Autophagy is a very important and highly regulated cellular process, through which damaged organelles and proteins are degraded by the lysosome. It facilitates nutrient recycling in cells, and alterations in this pathway underlie the pathogenesis of several diseases. Autophagy was first described by Professor Yoshinori Ohsumi, who received the Nobel Prize in Medicine or Physiology in 2016 for the discovery. Currently, autophagy is one of the most consensual therapeutic targets for neurodegenerative diseases and cancer, both through gene therapy and pharmacological approaches. For this reason, the development of gene addition therapies to modulate autophagy is an important focus of research and interest in the gene therapy field. 6 122 6.3.1 The Autophagy Pathway Gene Therapy Strategies: Gene Augmentation LC3-I that conjugates with PE (involving Atg7 and Atg3), generating the LC3-II form. The end of this process completes the maturation step of autophagy, resulting in a mature autophagosome. The fusion of the autophagosome with the lysosome forms the autolysosome, where the engulfed substrates are degraded through acidification and lysosomal hydrolase activity, concluding the autophagy process. As a very dynamic, complex and important cellular pathway, several guidelines have been established to study and monitor the autophagy process [21, 22], wherein two important autophagy markers emerge: LC3-II and SQSTM1/p62 (Sequestosome 1). During autophagy, the LC3-II residing in the inner membrane of the autolysosome is degraded, whereas the LC3-II in the outer membrane is recycled. Consequently, a decrease in LC3-II levels is probably related to an increase in degradation and in autophagy levels. SQSTM1/p62 is an essential cargo receptor involved in selective autophagy, delivering polyubiquitinated cargoes to the autophagy pathway. It interacts with LC3 and is specifically degraded by autophagy; thus, its levels are also correlated with activation or inhibition of this autophagy. There are three known types of autophagy (Fig. 6.3): microautophagy, chaperone-mediated autophagy and macroautophagy. Microautophagy is a nonselective degradation pathway that involves the direct engulfment of cytosolic material through lysosomal invagination and its degradation in the lysosome [16]. Chaperone-mediated autophagy (CMA) is a selective degradation pathway in which proteins are targeted for degradation through a recognition motif in their amino acid sequences by chaperones. These selected proteins directly cross the lysosome membrane and enter its lumen for degradation [17]. Finally, macroautophagy, commonly simply referred to as autophagy, is characterized by the formation of double-­ membrane vesicles named autophagosomes that engulf cytoplasmic material and fuse with lysosomes, ultimately degrading that material [18]. Initially described as an apoptosis mechanism, autophagy is now recognized as a cellular survival mechanism, playing an essential role in cellular and energy homeostasis. Its cellular importance is highlighted by the evolutionary conservation found in the autophagy pathway, from yeast to mammals. Autophagy is highly regulated by different proteins that control the initiation, elongation, maturation and fusion steps of the process, 6.3.2 Autophagy Terms Glossary through mTOR-dependent and mTOR-­ independent pathways [19]. The initiation step of • Phagophore – double-membrane structure that starts the sequestering of the cytoplasmic autophagy starts with the phosphorylation of the cargo for degradation. The continuous elongaUnc51-like kinase (ULK) complex and the fortion of the phagophore leads to its complete mation of the phagophore. Additional proteins closing, forming the autophagosome. including several Atg autophagy-­ related (Atg) proteins and beclin-1 are also essential for phag- • Autophagosome – double-membrane comophore formation and autophagy initiation. The partment that contains the cytoplasmic matecurrent view postulates that several cellular comrial for degradation and that is formed by the partments contribute to the phagophore growth, expansion and closing of the phagophore. including the cellular membrane, the Golgi com- • Lysosome – membrane-enclosed organelles plex and the endoplasmic reticulum [20]. In the containing a wide range of enzyme capable of elongation step, two ubiquitin-­like systems are degrading different biomolecules, such as proneeded, the Atg12-­Atg5-­Atg16L1 complex, teins, lipids and carbohydrates. which dissociates after the autophagosome for- • Autolysosome – autophagic compartment mation, and a second system resulting from the resulting from the direct fusion of an autophaconjugation of microtubule-­associated protein 1 gosome with a lysosome. light chain 3 (LC3) and phosphatidylethanol- • Autophagolysosome – specific situation that amine (PE). LC3 is cleaved by Atg4B, yielding occurs during some types of xenophagy 6.3 Gene Addition to Modulate Autophagy Fig. 6.3 The different autophagy mechanisms. Microautophagy (1) is a degradation pathway that involves the direct engulfment of cytosolic material through lysosomal invagination. Chaperone-mediated autophagy (2) is a selective degradation pathway in which proteins are targeted for degradation by chaperones, through a recognition motif in their amino acid sequences. 123 Macroautophagy (3), commonly simply known as autophagy, starts with the formation of the phagophore that involves the cellular material to be degraded, which after closing forms double-membrane vesicles, named autophagosomes. These vesicles fuse with the lysosomes, forming the autolysosomes in which the material is degraded. 6 124 (selective autophagy that is used for eliminating invading pathogens), where there is fusion of a phagosome with a lysosome [23]. 6.3.3  pregulation of the U Autophagy Pathway as a Therapeutic Strategy for Machado-Joseph Disease/ Spinocerebellar Ataxia Type 3 Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) belongs to the group of polyglutamine (polyQ) diseases, which includes nine inherited incurable neurodegenerative disorders that are caused by an abnormal expansion of CAG trinucleotide repeats present in the coding regions of single, and otherwise unrelated, genes. MJD/SCA3 is an autosomal inherited disease caused by an unstable expansion of a CAG repeat sequence in the ATXN3 gene, which is translated into an abnormal polyQ tract within the ataxin-3 (atxn3) protein (Fig. 6.4) [24]. One important hallmark of MJD/SCA3 is the presence of intranuclear protein aggregates in neurons of selected regions of the central nervous system, especially the cerebellum, the brain stem, the basal ganglia, Fig. 6.4 ATXN3 gene and ataxin-3 protein structures, highlighting the CAG/polyglutamine repetition range within healthy individuals and the patients with Machado-­ Joseph disease (MJD). The ATXN3 genes includes 11 exons (E1-11) and the CAG repeat region is localized in Gene Therapy Strategies: Gene Augmentation some cranial nerves and the spinal cord. Until now there is no therapeutic option to delay or stop the disease progression, and therefore gene therapy arises as an important possibility to treat and even cure MJD/SCA3 patients [25]. The importance of autophagy in neurons becomes very clear when the knockout of key autophagy-related genes in animal models yielded a neurodegeneration phenotype very similar to the one observed in several neurodegenerative diseases [26]. Now it is consensual that defects in the autophagy pathway underlie the pathogenesis of different neurodegenerative diseases, including MJD/SCA3, where abnormal accumulation of several autophagy proteins was detected in patients’ samples and animal models [27, 28]. It is also consensual that targeting autophagy constitutes an effective therapeutic strategy for these diseases (using both gene therapy and pharmacological approaches), aiming to reduce the pathological aggregates and to improve neuronal homeostasis. Following this idea, two important preclinical studies show that a gene addition therapy strategy inducing the expression of the gene codifying beclin-­ 1 (BECN1) was able to reduce motor deficits and neuropathological abnormalities in different exon 10 (E10). The ataxin-3 protein is mainly composed of an N-terminal catalytic domain and a C-terminal tail with two or three ubiquitin-interacting motifs (UIMs) and the polyglutamine stretch.