Gene Therapy for Cancer PDF

9.2 Gene Therapy for Cancer d­ evelopment of suicide gene therapy (Fig. 9.2). The idea behind this strategy is to deliver into tumor cells a gene encoding a cytotoxic protein. However, currently, different other types of gene therapy targeting cancer are being investigated and tested. Overall, gene...

9.2 Gene Therapy for Cancer d­ evelopment of suicide gene therapy (Fig. 9.2). The idea behind this strategy is to deliver into tumor cells a gene encoding a cytotoxic protein. However, currently, different other types of gene therapy targeting cancer are being investigated and tested. Overall, gene therapy for cancer employs one of four strategies [3]: (1) introduction of suicide genes, which induce the generation of compounds that are toxic to tumor cells; (2) induction of cell lysis using modified viruses; (3) introduction of immunomodulatory genes, aiming to induce or increase the immune system response; and (4) introduction of tumor-­ suppressor genes, which will block cell division. Of course, this simple classification of gene therapy strategies targeting cancer might be reductive, as cancer is a very complex disease. Furthermore, this general strategies hide the fact that different approaches are strongly interconnected, and one particular approach may, for example, cause cell lysis and induce the immune system response at the same time. Cancer is one of the main targets for gene therapy, and advances in this field led to the approval of Imlygic®, which is a suicide gene therapy product for melanoma treatment. It is based on the intratumoral delivery of a weakened form of herpes simplex virus type 1 (e.g., the neurovirulence factor ICP34.5 was deleted) that can infect and multiply inside melanoma cells, killing them; moreover, it makes the infected cells produce the human cytokine granulocyte macrophage colony-stimulating factor gene (GM-CSF), which stimulates the patient’s immune system to destroy the cancer cells. The therapy, now approved both in Europe and the USA, is able to selectively recognize and destroy Fig. 9.2 Overview of suicide gene therapy targeting cancer, aiming to cause cell death through the introduction of a toxic gene. 167 malignant cells with a minimal effect on normal cells [4]. 9.2.1 Suicide Gene Therapy Suicide gene therapy can be achieved using at least two strategies [5]: an indirect form in which gene therapy is accomplished through an enzyme-­ activated prodrug that will be converted into a lethal drug inside the cells, or the direct delivery of proapoptotic genes (Fig. 9.3). Importantly, both forms aim to produce cell death without affecting normal cells. The first strategy uses genes that codify for enzymes that convert a prodrug (which is afterward administrated) into a compound that is actively toxic to the cell, leading to its death. One of the most popular systems uses the herpes simplex virus thymidine kinase gene (HSV-tk), which converts the antiviral drug ganciclovir (GCV) to monophosphate, that is then metabolized by cell kinases into GCV-­ triphosphate, inhibiting DNA synthesis and leading to cell death [6]. This suicide gene therapy strategy also has the advantage of the bystander effect, which is a potentiation of the effect whereby the prodrug efficacy will be extended to neighboring cells. Other popular systems employ the enzyme cytosine deaminase and the prodrug 5-flurocytosine (5-FC) or the cytochrome P450 and the prodrugs cyclophosphamide (CPA) and ifosfamide (IFO). The other type of suicide gene therapy is based on the introduction of proapoptotic genes. For example, the delivery of the gene codifying wildtype p53 (which is frequently mutated in several 168 Fig. 9.3 The different types of gene therapy strategies targeting cancer. Suicide gene therapy can be achieved using at least two strategies, an indirect form in which gene therapy is accomplished through an enzyme-­ activated prodrug that will be converted into a lethal drug in cells, or a direct form consisting on the delivery of proapoptotic genes (1). The idea of oncolytic gene therapy is to apply viruses directly into the tumor, and the patho- 9 Gene Therapy Applications genic agents will induce tumor cell lysis (2). In the immunomodulatory gene therapy, there is the introduction of a gene leading to the stimulation or enhancement of antitumor immunity (3). In the tumor-suppressor gene therapy, there is the introduction of tumor-suppressor genes as a way to fight cancer, preventing uncontrolled cellular growth (4). 9.2 Gene Therapy for Cancer human tumors) can restore the normal function of the protein and promote cellular apoptosis in tumors. In fact, Gendicine®, the first gene therapy product approved in China, is based on this strategy, delivering wild-type p53 gene through adenoviral vectors. The use of other genes aiming to promote cell death is also under investigation, including the genes codifying caspase 3 and Smac. 9.2.2 Oncolytic Gene Therapy Besides the important use of viruses as carriers, their natural infectious features may also be used for cancer gene therapy applications. The idea of oncolytic gene therapy is to apply viruses directly into the tumor, where the pathogenic agents will induce tumor cell lysis (Fig. 9.3). These so-called oncolytic viruses can lead to cellular death through oncolysis, destruction of tumor blood vessels, antitumor immune activation, or even through the promotion of the expression of therapeutic genes in parallel with the lysis effect. For example, attenuated forms of herpes simplex virus type 1 (HSV-1) only able to replicate in tumor cells can be introduced into these cells leading to lysis upon infection. This strategy presents two important features. First, there is a potentiation effect, since the new viruses produced (these are replicative viruses) are able to infect neighboring tumor cells. Second, the use of these oncolytic viruses and their replication will promote an immune response, thus enhancing the therapeutic effect. This strategy is the basis of Imlygic®, an HSV-1-based system that included several genome modifications, such as deletion of ICP34.5 and ICP47 genes and the insertion of a transgene encoding GM-CSF, as previously explained. Moreover, the insertion of cellular promoters for tumor-targeted replication was also used to increase tumor specificity [7]. In China, another oncolytic gene therapy named Oncorine (H101) was also approved. Oncorine is an oncolytic adenovirus encoding the p53 protein, to be used in combination with chemotherapy for the treatment of nasopharyngeal carcinoma [8]. Other viruses are also being inves- 169 tigated in clinical trials as oncolytic gene therapy strategy effectors, namely vaccinia virus, reovirus or measles virus [9]. 9.2.3 Immunomodulatory Gene Therapy The idea behind immunomodulatory gene therapy is the recognition that growing tumors actively evade the immune system and that stimulation or enhancement of antitumor immunity can be a therapeutic strategy to fight cancer. In fact, the 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison and Tasuku Honjo for their discovery of cancer therapy by inhibition of negative immune regulation. In a gene therapy setting, this strategy can take advantage of the enhancement of the immune response through the delivery of proinflammatory genes (Fig. 9.3). Furthermore, the use of modified cells (e.g., T-lymphocytes or dendritic cells) is another strategy for immunomodulatory therapy, thus combining gene and cell therapy [10]. For example, the combination of an immunological checkpoint blockade with CAVTAK™, which is an oncolytic coxsackievirus, led to a very good response and disease control rates in a phase Ib clinical trial (NCT02307149) for advanced melanoma [11]. Also, preclinical studies showed that the combination of oncolytic virus (expressing C-X-C motif of chemokine 11 precursor) and immunological checkpoint blockade reduced tumor immunosuppressive activity and boosted T-cell infiltration in the tumor [12]. 9.2.4 Tumor-Suppressor Gene Therapy The introduction of tumor-suppressor genes as a way to fight cancer is a particular case of suicide gene therapy, as most cancer patients present mutations or even deletions in tumor-suppressor genes. The main goal of this gene therapy strategy is the functional restoration of those mutated/ deleted genes and prevention of the uncontrolled cellular growth (Fig. 9.3). Additionally, this 9 170 group of strategies includes therapies targeting angiogenesis, aiming to cut off the nutrient supply to tumors, thus preventing their growth and contributing to their shrinkage. As well as for suicide gene therapy, the use of the p53-codifying gene, or several downstream mediators, can help to prevent tumor growth [13]. Additionally, preclinical studies show that the transfer of genes encoding cyclin inhibitors, and even microRNAs, could be used in this strategy. This type of strategies also includes the blocking of oncogenes expression through gene silencing approaches (based on RNAi or ASOs) and the expression of genes controlling checkpoints of the cell cycle, trying to stop cellular division. 9.2.5 Comparing Gene Therapy Strategies for Cancer Along with rare neurodegenerative diseases and eye-affecting conditions, gene therapy strategies targeting cancer are probably the most advanced in terms of preclinical and clinical research. Like it was mentioned, several of the described strategies are used together and take advantage of different features both of the delivered gene and Gene Therapy Applications the delivery vector. As they present advantages and disadvantages, choosing between particular strategies should be based on the application purpose and the type of tumor, among other considerations (Table 9.1). Moreover, gene therapy strategies for cancer may also be used in combination with conventional treatments such as chemotherapy or radiotherapy, which is another important point that should be considered in the definition of the strategy to study. 9.2.6 Challenges to Gene Therapy Targeting Cancer As mentioned throughout this book, several conditions must be addressed and considered to ensure the success of a gene therapy approach, regardless of the target disease. However, gene therapy targeting cancer has some particularities that should also be taken into consideration. In the first line of obstacles or challenges to tackle is the heterogeneity of cancers, ranging from the anatomical differences to the individual differences among the same type of cancer and even to the heterogeneity of the cell types whithin a cancer. All these features make the application Table 9.1 Main advantages and disadvantages of the different strategies of gene therapy targeting cancer. Gene therapy strategy Suicide gene therapy Oncolytic gene therapy Immunomodulatory gene therapy Tumor-suppressor gene therapy Advantages Proved safe in clinical trials Action potentiation – bystander effect May be used in combination with conventional therapies Replication only in tumor cells Action potentiation – bystander effect May be used in combination with conventional therapies May enhance the action of the immune system Promotion of long-term immunity Good efficacy results in vivo May be used in combination with conventional therapies Proved safe in clinical trials May be used in combination with conventional therapies Disadvantages Difficulty in targeting all tumor cells Low efficacy of gene transfer in vivo Viruses may be cleared by the immune system Low efficacy of gene transfer in vivo Must overcome tumor-induced immunosuppression Autoimmune side effects Existence of redundant pathways in tumors Low efficacy of gene transfer in vivo 9.3 Gene Therapy for Eye Conditions of gene therapies (and also of conventional therapies) very difficult, bringing with them a high degree of variation in their efficacy. Another crucial aspect is the ability to specifically target tumor cells, especially when using oncolytic suicide gene therapy. One way to try to ensure specificity is targeting the vectors to specific receptors of tumor cells, or using promoters specific to those cells, allowing the preservation of normal, non-diseased, cells. The safety profile of vectors is another aspect that must me guaranteed, especially in the gene therapy strategies that use replicative viral vectors. Safety can be improved also by engineering the viral particles so as to specifically infect the tumor cells. Another major concern of gene therapy applied to cancer is that efficacy of several strategies was shown to be reduced due to the presence of circulating antibodies, which is particularly relevant when using viruses as a delivery vector. Despite all these and other challenges, the future of gene therapy for cancer seems bright, with the prediction that several products presently in advanced stages of development could be approved for marketing in the next years (Table 9.2). 9.3 Gene Therapy for Eye Conditions Since the beginning of gene therapy, the eye and more specifically the retina was considered a privileged target organ for the development of new therapeutics. Briefly, the eye comprises two main regions: the anterior part (with the cornea, lens and conjunctiva) and the posterior part, with the retina as the prominent structure. The retina is considered part of the brain, consisting of different cellular layers such as ganglion cells, nerve fibers, light-sensing photoreceptors and the retinal pigment epithelium (RPE), among others. Despite being a highly specialized structure, compared to other organs or tissue, the retina has several advantages that make it very suitable for gene therapy. First, it can be easily and directly accessed, which allows direct and accurate delivery of the therapy. Moreover, as a closed system, 171 the delivery of genes is limited to the eye structures, without propagation into peripheral organs. Second, the retina allows an easy visualization, which permits a noninvasive follow-up of the intervention throughout time. Third, the eye is an immunologically privileged site, which limits the immune response to the gene delivery. Finally, there are two eyes, allowing the possibility of maintaining an untreated contralateral eye, as a valuable control for the disease natural history, to assess the treatment efficacy and to evaluate its safety profile. Because of all these advantageous features, there is a wide range of preclinical and clinical studies evaluating different gene therapy approaches for several retinal diseases. It is thus also not surprising that one of the currently approved gene therapy products targets a retinal disease. Luxturna™ (Spark Therapeutics) is a gene therapy product consisting of AAV vectors that mediate delivery of a normal copy of the RPE65 gene, administered through subretinal injection, to treat an inherited retinal disease caused by mutations in both copies of the aforementioned gene. 9.3.1  Privileged Immunologic A Organ One of the important obstacles to the success of gene therapy is the immune response, which is particularly relevant when genes are delivered by viral vectors. Several features are present in the eye to maintain its immune privilege [14], circumventing the immune response and making them a very good target for gene therapy. First, several physical barriers (such as the blood-retina barrier and the lack of efferent lymphatic vessels) prevent the free exchange of cells and large molecules between the eye and the rest of the organism. Second, the ocular microenvironment inhibits the immune-competent cells, due to the production of several factors (e.g., TGF-β) and the direct action of ocular cells (e.g., retinal glial Muller cells). Third, the eye is able to regulate the immune response directly. All these properties of the eye and the retina constitute advantages that make them good targets for gene therapy strategies. In vivo Retroviral vector DLBCL – Diffuse large B-cell lymphoma GM – CSF - granulocyte macrophage colony-­stimulating factor MM – Multiple myeloma CAR – chimeric antigen receptor NMIBC – Non-muscle invasive bladder cancer TK – thymidine kinase MIBC – Muscle invasive bladder cancer BCG – Bacillus Calmette-Guérin NSCLC – Non-small cell lung cancer Toca 511 (vocimagene amiretrorepvec) Prostate cancer/hepatocellular carcinoma, pancreatic adenocarcinoma/NSCLC Recurrent high-grade glioma Adenovirus In vivo Nadofaragene firadenovec/Syn3, rAd-IFN/Syn3 (Instiladrin) ProstAtak Adenovirus In vivo E10A In vivo Relapsed or refractory MM NMIBC/MIBC T-cells Adenovirus Ex vivo In vivo Squamous cell carcinoma of the head and neck BCG-unresponsive NMIBC Relapsed or refractory DLBCL Ex vivo Adenovirus CAR T-cells Hepatocellular carcinoma Vaccinia virus T-cells In vivo Suicide + immunomodulatory Suicide + immunomodulatory TK + valacyclovir (drug) Cytosine deaminase +5-FU (drug) Immunomodulatory Immunomodulatory Oncolytic + immunomodulatory Tumor-suppressor Therapy mechanism Oncolytic + immunomodulatory Tumor-suppressor Oncolytic + immunomodulatory Oncolytic + immunomodulatory Immunomodulatory Interferon alfa-2b Endostatin CAR T-cells GM-CSF CPI (PD1 inhibitor) VBL’s PPE-1-3x proprietary promoter GM-CSF Mesothelioma Glioblastoma Adenovirus Adenovirus In vivo In vivo Name DNX-2401 (tasadenoturev) ONCOS-102 VB-111 (ofranergene obadenovec) Pexa-Vec (pexastimogene devacirepvec, JX-594) JCAR017 (lisocabtagene maraleucel) bb2121 CG0070 Therapeutic gene/cell Modified Ad genome Indication Glioblastoma/gliosarcoma Vector Adenovirus Type of gene therapy In vivo Table 9.2 Gene therapies targeting cancer that are in the final stages for marketing approval. II/III III III III I/II II I III I/II III Clinical study phase II Tocagen Advantagene FKD Therapies, Ferring Pharmaceuticals Marsala Biotech Juno Therapeutics, Celgene bluebird bio, Celgene Cold Genesys SillaJen Targovax VBL Therapeutics Manufacturer DNAtrix 172 9 Gene Therapy Applications 9.3 Gene Therapy for Eye Conditions 9.3.2 173  ene Delivery Routes G and Vectors The delivery of genes to the retina takes advantage of the extensive research made concerning the pharmacokinetic profiles of conventional drugs and the multiplicity of routes and types of administration tested [15]. Nevertheless, despite the variety of delivery routes that could be used, due to efficacy issues, gene delivery is mainly performed by intravitreal or subretinal injection. The latter route leads to more rapid expression of the transgene compared to intravitreal injection. However, both routes also have limitations and barriers that need to be surpassed. For example, the genes delivered by subretinal injection need to overcome the external membrane of the photoreceptors. On the other hand, the intravitreal route dilutes the genes in the vitreous and has to overcome the inner limiting membrane [16]. Systemic administration, in the blood circulation, of recombinant AAV targeting the eye was also tested in different studies, as it is a less invasive and challenging pathway. However, this route needs high-titer injections of viral vectors and could potentially lead to off-target toxicity effects. Concerning the delivery vehicle, several studies have used non-viral vectors to deliver genes to the eye (Table 9.3). However, these methods frequently lack efficiency, and some of them are clinically inviable due to ocular complications related to their application. Therefore, the use of recombinant viruses as vectors to deliver a gene directly to the eye rapidly increased in preclinical and clinical studies. Out of all the possibilities, AAV emerges as the preferred vector, due to their low immunogenic profile, the stable transgene expression they induce and the natural tropism of specific serotypes to eye cells. A huge effort is continuously being made to improve AAV efficacy, including optimization of the viral transduction or of the specificity of viral tropism. For example, to overcome the main limitation of AAV vectors, which is the limited cloning capacity, “overstuffed” and dual AAV vectors [17] were developed (see Chap. 3). However, despite being very useful for large transgenes, the applicability of dual AAV vectors is currently limited, because the system requires the co-expression of two vectors in the same cells, and the subsequent occurrence of homologous recombination. The simultaneous success of both steps is needed, which has been limiting the efficiency of dual vectors in gene therapy. 9.3.3  ene Therapy Trials for Ocular G Conditions The heterogeneity of conditions affecting the eye and of the genes and cells involved is reflected by the variety of gene therapy trials developed so far, using a multiplicity of vectors, genes and administration routes. Nevertheless, currently, only 1.4% of gene therapy trials have targeted Table 9.3 Examples of gene therapy studies targeting the retina using different delivery methods. Physical methods Chemical methods Method Naked DNA Gene VEGF Model CD-1 mice Electroporation sFlt-1 Gene gun Ultrasounds GFP GFP Liposomes Polymers Rpe65 Oligonucleotides Albino Lewis rats Rabbits New Zealand albino rabbits Blind mice Lewis female rats Route of administration Intrastromal corneal injection Injection in the suprachoroidal space Cornea Intravitreal Subretinal injection Intravitreal injection References Stechschulte et al. [34] Touchard et al. [35] Tanelian et al. [36] Sonoda et al. [37] Rajala et al. [38] Gomes dos Santos et al. [39] 9 174 ocular diseases. From these, most of the clinical studies have focused on the RPE65 gene, ­especially for treating Leber’s congenital amaurosis (LCA). Several strategies were also studied for other ocular conditions, such as choroideremia, achromatopsia or retinitis pigmentosa. In Table 9.4, selected gene therapy clinical trials targeting eye conditions are shown, providing an overview of the different target conditions, strategies and vectors used. 9.3.4 Challenges to Gene Therapy Targeting the Eye The current gene therapy tools targeting the retina are well improved and developed. There is a wide range of delivery routes, several vectors, and diverse gene transfer reagents available, and there is data from animal studies on the safety profiles of gene therapy strategies, as well as extensive data on their immune response. Nevertheless, the increasing accuracy in diagnosing retinal diseases has led to the discovery of more than 250 different genetically distinct retinal diseases that are now known [18] and being curated in a public database (https://sph.uth.edu/ RETNET/). This number highlights the high diversity of retinal diseases-causing mutations, and suggests the consequent, difficulty in developing gene therapy products for each one of them. Another important challenge that gene therapy targeting the retina needs to overcome is the difficult transition of the preclinical animal results to human studies. Contrary to other targets, the human eye is far more specialized than that of current rodent models and, for this reason, several studies need to be performed in species with a more complex eye, like nonhuman primates. Of course, this raises ethical and economic issues that need to be carefully considered and discussed. Additionally, it is particularly important to develop vectors specifically directed to the human retina, as frequently the transduction pattern is different in rodents compared to humans or larger animals. 9.4 Gene Therapy Applications Gene Therapy for Cardiovascular Diseases (CVDs) Cardiovascular diseases (CVDs) constitute a serious health problem, being the major cause of mortality and morbidity worldwide, and their prevalence is still increasing. CVDs comprise a group of disorders of the heart and blood vessels, for example, coronary heart disease, cerebrovascular disease and peripheral arterial disease, among others. Heart attacks and strokes, despite usually corresponding to acute events, are also classified as CVDs. There has been a huge advance in the pharmacological and surgical therapies for CVDs, which resulted in symptom mitigation and in the reduction of disease progression; however, there is still a lack of effective therapies to effectively cure and treat CVDs. Therefore, gene therapy appears as a promising strategy for the treatment of both inherited and acquired CVDs gene. The potential of gene therapy in the context of CVDs became very clear a long time ago when direct intra-arterial gene transfer was performed using endovascular catheter techniques [19]. In fact, the multiplicity of surgical techniques used for the different CVDs contributed to the extensive gene therapy trials performed for these diseases, as several administration routes have already been tested and established (Table 9.5). In terms of vectors, the first attempts used non-viral methods; however, similarly to other applications, viral vectors have become the preferable system in CVD studies. In this group, there are studies using adenoviral vectors, AAV vectors and lentiviral vectors. In the case of AAV vectors, serotypes 1, 6, 8 and 9 have been identified to have a good tropism for cardiac tissue, after systemic delivery. Another important issue regarding gene therapy studies for CDVs is the therapeutic genes to transfer. The most promising results were obtained with genes that induce angiogenesis or vasculogenesis or genes that encode for proteins involved in the cardiomyocytes Ca2+ pathway (Table 9.6). Nevertheless, several other genes were also studied [20], although until now no gene therapy product has been approved. 9.5 Gene Therapy for Neurodegenerative Diseases 175 Table 9.4 Selected gene therapy clinical trials targeting eye diseases. Therapeutic gene Phase RPE65 III Condition Leber’s congenital amaurosis Choroideremia CHM Achromatopsia Retinitis pigmentosa Stargardt disease Neovascular age-related macular degeneration CNGB3 PDE6B ABCA4 AntiVEGF protein Vector AAV2 Route of administration Subretinal III AAV2 Subretinal I/II I/II I/II I/II AAV8 AAV2/5 Lentivirus AAV8 Subretinal Subretinal Subretinal Subretinal Therefore, more studies are needed both at the preclinical and clinical levels to deliver a safe and efficient gene therapy product for CVDs. 9.5 Gene Therapy for Neurodegenerative Diseases The brain and its molecular networks and cellular circuits are quite complex, which complicates its study in normal and pathological conditions. Neurodegenerative diseases are defined by a progressive dysfunction of the nervous system, which is normally translated into severe symptoms and a debilitating phenotype. Normally, these are lateonset diseases, affecting a large part of the world population, which is a reflex of an increased elderly population worldwide. Therefore, they represent a huge burden for current societies, without any therapy available to stop or delay disease progression. In fact, current therapies are only symptomatic, with very limited effects and do not target the underlying molecular mechanisms of pathogenesis and degeneration. The difficulty in treating neurodegenerative diseases has several causes; however, their complex nature and the limited accessibility of the central nervous system (CNS) are probably the two most important ones. First, neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s diseases are mainly sporadic, without a clear cause, which strongly limits the ability to rationally develop therapies for them. Even monogenic Sponsor Spark Therapeutics Nightstar Therapeutics MeiraGTx Horama S.A. Sanofi REGENXBIO Inc. Trial ID NCT00999609 NCT03496012 NCT03001310 NCT03328130 NCT01367444 NCT03066258 neurodegenerative diseases, like the polyglutamine diseases, have a complex molecular pathogenesis that only now is starting to be completely elucidated. The other important point is the existence of the blood-brain barrier (BBB), preventing the access of many drugs to the CNS, which is the part of the nervous system that is most commonly affected in these diseases. Furthermore, there is a high number of different neurodegenerative diseases, and each one is characterized by a considerable degree of heterogeneity and by a variable incidence; combined, these aspects constitute additional challenges to the search for therapeutic solutions. Two other important factors contribute to the lack of efficient therapies: the CNS has a poor ability to repair neuronal damage, which strongly limits the success of a therapy in actually reverting neuronal loss, and the evaluation of the clinical outcomes after a therapy administration is extremely difficult, due to the heterogeneous progression of the neurodegenerative process and to the lack of established biomarkers. Gene therapy arises as a powerful tool to treat neurodegenerative diseases, trying to restore missing functions or to improve neuronal homeostasis, or ultimately to enact the correction of the molecular pathogenesis. The potential of gene therapy in the treatment of n­ eurodegenerative diseases was already highlighted throughout this book, for example, for spinal muscular atrophy (SMA). However, even if promising, there is still a long way to be traveled in order to increase the number of gene therapy solutions for the different neurodegenerative diseases. 9 176 Gene Therapy Applications Table 9.5 Advantages and disadvantages of different gene administration methods targeting cardiovascular diseases. Gene administration method Direct intramyocardial injection Anterograde arterial infusion Retrograde intravenous infusion Pericardial delivery Aortic cross-clamp left ventricular cavity infusion 9.5.1 Advantages Decreased risk of immune response High density of gene transfer, limited to the cardiac tissue Simple and safe procedure Simple and minimally invasive procedure with selectivity for the cardiac tissue Can lead to a homogenous distribution of the gene Can lead to high levels of transduction in the cardiac cells Can lead to a homogenous distribution of the gene Can increase the transduction levels Safe and minimally invasive Increased gene transfer efficiency Administration Routes to the CNS The choice of the administration route is an essential point for all gene therapy strategies, but it is especially critical in the context of neurodegenerative diseases due to the presence of the BBB. To overcome this barrier, the strategy most commonly used so far has been the local and direct delivery of the therapeutic gene into the brain parenchyma, through neurosurgical stereotaxic injections. However, an important limitation of the direct intracranial injection is the associated local trauma, as well as the stimulation of inflammation and toxicity-inducing events. Also, the procedure is associated with a poor spread of the vector, limited around the injection site. Of course, this limitation can be overcome with multiple injections, although this strongly increases the complexity of the surgery and its potential side effects. Additionally, the procedure is preferably performed only once due to the risks to the patient, thus precluding multiple administrations at different time points. An alternative to this procedure can be the delivery of the vectors with the therapeutic gene into the cerebrospinal fluid (CSF), which can be Disadvantages Expression of the gene limited to the local of the injection Multiple injections are needed Potential damage from the needle May be inefficient Can result in the systemic delivery of the transgene Potential risk of ischemic events May be inefficient Transgene expression limited to superficial epicardium High risk of myocardial injury Not cardiac specific Requires open chest surgery accessed through a different route. This alternative will increase the spread of the vector, although it has an important degree of invasiveness and complexity in some of its routes. Maybe the one exception is the intrathecal injection, which is a common and relatively safe procedure in current medical practice. However, all these routes are relatively complex, and therefore noninvasive and peripherical routes of gene delivery were also developed and studied. For motor neuron diseases, an interesting route consists on the intramuscular injection of the vectors, which is a minimally invasive procedure that could reach the CNS by retrograde transport through the motor neurons. However, the least invasive and probably most straightforward delivery route is the systemic administration of the vectors, especially through intravenous injection. This method has the advantage of being safe, allowing repeated administrations and the introduction of high amounts of vectors, if needed. However, the BBB limits the access of the systemic molecules to the CNS, and therefore the vector must have the ability to cross it. To achieve this, several studies have reported non-viral vectors coupled with molecules that facilitate or mediate the BBB crossing. On the Severe and diffuse triple vessel coronary disease Hypoperfused area of viable ventricular muscle Refractory coronary artery disease Severe stable ischemic heart disease Coronary heart disease Chronic stable angina Condition Coronary heart disease Therapeutic gene FGF2 (fibroblast growth factor 2) FGF4 (fibroblast growth factor 4) VEGF-A (vascular endothelial growth factor A) VEGF-A (vascular endothelial growth factor A) HGF (Hepatocyte growth factor) HIF1-alpha (Hypoxia-­ inducible factor 1-alpha) VEGF+FGF Adenovirus/ liposome Naked plasmid DNA Phase II Phase II Adenovirus Adenovirus Naked plasmid DNA Phase I Phase I Phase II Phase I/II Vector Naked plasmid DNA Adenovirus Development stage Phase II Table 9.6 Selected gene therapy clinical trials targeting cardiovascular diseases. Intramyocardial injections Percutaneous intramyocardial injection Intracoronary infusion Direct intramyocardial injection Intracoronary infusion Intracoronary infusion Delivery method Intracoronary infusion Improved exercise tolerance 52 No demonstrated improvement in cardiac perfusion No safety concerns No adverse events No improvements in myocardial perfusion No major adverse events Findings No improvements in myocardial perfusion No major adverse events 13 18 80 103 79 Number of patients 337 9.5 Gene Therapy for Neurodegenerative Diseases 177 9 178 side of viral vectors, the discovery of the natural ability of AAV9 to bypass the BBB opened a new opportunity to treat neurodegenerative disease with viral vector-­mediated gene therapy. 9.5.2 Candidate Conditions for Gene Therapy Neurodegenerative disease is a broad term that includes several chronic conditions that cause a slow, progressive and irreversible loss of neurons. The brain regions and neuronal subtypes affected are different among these diseases, which translates into different phenotypes and symptoms. This group of diseases includes Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and other motor neuron diseases such as spinal muscular atrophy (SMA), and the polyglutamine diseases. This latter group constitutes the larger group of monogenic neurodegenerative diseases and comprises Huntington’s diseases (HD), six forms of spinocerebellar ataxia (SCA1, 2, 3, 6, 7 and 17), dentatorubral-pallidoluysian atrophy (DRPLA), and spinal and bulbar muscular atrophy (SBMA). Aside from these chronic neurodegenerative diseases, other acute conditions affecting the CNS, such as stroke and spinal cord injury, can also be targeted by gene and cell therapy strategies. 9.5.3  ectors for Delivering Genes V into the CNS Like for any other organ or tissue, the ideal vector used to deliver genes into the CNS should have specifically appropriate features, most of which have already been discussed throughout this book. Nonetheless, taking into consideration the important limitations pointed above, some of the conditions that are crucial for the success of gene therapy targeting the brain deserve a closer look. First, the gene introduced must be delivered efficiently to the brain. Second, since most of the neurodegenerative diseases result from a malfunction of specific regions and neuronal popula- Gene Therapy Applications tions of the brain, the gene should be preferentially delivered to the affected neurons, avoiding the other cells of the CNS. The third important consideration concerns the levels and duration of gene expression. The access to the CNS is difficult; thus, ideally, gene delivery should be performed once, ensuring a therapeutic expression without cytotoxic side effects. To meet all these conditions and overcome the particularities of the CNS, most of the studies use viral vectors to deliver genes. The herpes simplex virus type 1 (HSV-1) vector has a natural tropism to neurons, which makes it a good vector targeting the brain. However, its high cytotoxic profile makes it unsuitable for most of the neurodegenerative diseases. Currently, most of the gene therapy applications using HSV-1 vectors aim to take advantage of this feature to treat glioblastomas, which are brain tumors with a very poor prognosis. Even more limited is the use of adenoviral vectors for gene delivery to the brain, due to the strong immune response they elicit and their potential toxicity. On the other hand, lentiviral vectors are attractive as gene delivery systems for the brain, allowing a good tropism to neurons or glial cells, depending on the envelope pseudotyping. One of the main advantages of lentiviral vectors is their ability to integrate the transgene into the host cell genome, facilitating the goal of one-time therapy. However, the possibility of insertional mutagenesis, along with their HIV-1 origin, constitute important safety limitations to their use in the treatment of neurodegenerative conditions. Due to the limitations of both HSV-1 and lentiviral vectors, AAVs have emerged as good delivery vectors for the brain. AAVs can transduce nondividing cells and have the ability to confer long-term expression of the therapeutic gene without inflammation or toxicity. Moreover, the discovery of an AAV serotype that crosses the BBB provided an important boost to their use as vectors targeting the CNS. However, the use of AAVs is still limited, due to the presence of circulating preexisting antibodies against several AAV serotypes. Moreover, their small cloning capacity constitutes another important disadvantage. These considerations demonstrate that there is not a singularly perfect viral vector to deliver genes 9.5 Gene Therapy for Neurodegenerative Diseases to the brain; however, current advances in lentiviral and AAV vectors make them the most probable and most common choice in both preclinical and clinical studies. In the next sections, several gene therapy strategies for different neurodegenerative diseases are described, although diverse examples have already been mentioned and discussed in other chapters throughout the book. 9.5.4 Alzheimer’s Disease Alzheimer’s disease (AD) is the most common neurodegenerative disorder worldwide. The disease is mainly characterized by impairments in memory and behavior, and there are no therapeutic solutions available to stop the disease progression. Neuropathologically, it is characterized by a degeneration of cholinergic neurons, the formation of extracellular plaques of amyloid β peptide and the assembly of neurofibrillary tangles due to the accumulation of hyperphosphorylated tau protein. Despite being very common, less than 10% of AD cases are familial and, therefore, the intricate etiology of the disease complicates the development of therapeutic strategies. Several gene therapy strategies have been tested in preclinical studies; however, the clinical studies focused mainly on the expression of neurotrophic factors, especially the nerve growth factor (NGF) as a way to improve AD-associated deficits. A pioneering study showed that an ex vivo gene therapy approach, based on the administration of fibroblasts expressing NGF into the brain, was able to increase the density of cholinergic neurons in aged monkeys [21]. This study was the basis for a phase I clinical trial developed in the USA in eight patients with early-stage AD. In this study, autologous fibroblasts modified by retroviral vectors to produce and secrete NGF [22] were transplanted into the nucleus basalis of Meynert of the patients. A 22-month follow-up of six of the patients showed no adverse effects, a reduction in the speed of the cognitive decline of around 50%, and a significant increase in cerebral metabolism, evaluated by positron emission tomography (PET) scan. Moreover, a 179 brain autopsy of one of the patients showed robust expression of NGF and a dense concentration of cholinergic axons in the fibroblast graft. Although the results were promising, the high costs and complexity of the procedure prevented further development and the continuation of the studies in subsequent clinical trials. More recently, a phase I clinical trial injected NGF-codifying AAV2 particles into the basal forebrain of 10 AD patients [23], upon stereotaxic surgery. A follow-­up of 2 years showed that this in vivo gene therapy approach was well tolerated and was able to produce long-term expression of NGF. These promising results were the basis for a subsequent phase II clinical trial (NCT00876863). The first results of the study were somehow mixed, and 2 years after the procedure both groups showed a similar decline in cognitive function. Therefore, in 2015, Sangamo Therapeutics ended the development of this strategy, which was named CERE-110. 9.5.5 Parkinson’s Disease Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, arising as a heritable condition (in 5–10% of the cases) or, more commonly, as a sporadic disorder [24]. From a neuropathological point of view, it is characterized by neuronal loss in the substantia nigra, which causes striatal depletion of dopamine and the formation of intracellular inclusions of α-synuclein. Traditionally, motor symptoms associated with PD (e.g., rigidity, resting tremor) are mitigated by the reposition of dopamine levels with drugs such as levodopa. This treatment is able to counter some motor deficits; however, with disease progression, levodopa becomes less efficient. Therefore, there is an opportunity for gene therapy strategies and, in fact, several different approaches have been explored both in preclinical and clinical studies. There are several gene therapy studies using nonhuman primates focusing on normalizing either the dopamine production or the abnormal firing in the basal ganglia, while other strate- 9 180 gies aim to stop, or revert, neuronal loss [25]. In the same line, there are also several clinical trials performed in PD patients using gene therapy strategies (Table 9.7). Most of them were phase I clinical trials that passed the safety criteria and demonstrated some efficacy. However, in subsequent trials, almost all of them failed to show more efficiency than the placebo. One exception was a phase II clinical trial based on the local injection of the GAD (glutamic acid decarboxylase) gene, delivered by AAV2 into the subthalamic nucleus in 22 PD patients [26]. There was an improvement in the AAV2GAD-treated group compared with the control group; however, the effect was not better than the current standard of treatment, and therefore the development of the therapy was not continued. Another phase II clinical trial used AAV2 to deliver the neurturin (NRTN) gene into the putamen of 38 PD patients. Some encouraging results were observed, namely an increase of neurturin expression at the injection site, although its levels in the substantia nigra were not altered. Based on these results, a further phase IIb clinical trial was developed injecting the therapy both in the putamen and in the substantia nigra. However, there was no significant improvement in the clinical outcome of treated patients, and therefore the development of the therapy (named CERE-120) was not continued. Another interesting strategy was developed by Oxford Biomedica, using a polycistronic lentiviral vector to express three enzymes essential for dopamine synthesis: tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase I (GCH1). The therapy (named ProSavin) was tested in a phase I/II clinical trial targeting the sensorimotor part of the striatum and the putamen. The therapy proved to be safe, and the majority of the treated patients displayed some improvements in motor behavior [27]. The company is now preparing a phase I/IIa clinical trial with an improved version of the therapy named OXB-102. The results of these and other clinical studies highlight the complexity of developing gene therapies for PD. However, the relevance of the disease and the Gene Therapy Applications continuous development of strategies in preclinical studies promise more clinical trials with gene therapies in the future. 9.5.6  ysosomal Storage Diseases L (LSDs) Lysosomal storage diseases (LSDs) comprise a group of more than 50 different inherited metabolic diseases that result from a dysfunction of the lysosome, which leads to the accumulation of undigested or partially digested macromolecules. Despite being a metabolic diseases, around 70% of LSDs affect the CNS, leading to selective neurodegeneration in several brain regions and extensive neuroinflammation. LSDs are good candidates for gene therapy, as most of these diseases are autosomal recessive monogenic conditions. Therefore, there are several examples of preclinical and clinical studies using gene therapy for these diseases [28], including some strategies in the late stages of clinical development, which suggests that some gene therapies for LSDs may be approved in the near future. Metachromatic leukodystrophy (MLD) is caused by a deficiency in arylsulfatase A (ARSA), leading to an accumulation of sulfatides and demyelination of the CNS and peripheral nervous system, which translates into severe motor and cognitive defects. In 2013, an ex vivo gene therapy clinical trial study was performed in three patients with presymptomatic MLD. Autologous hematopoietic stem cells (HSCs) were treated with a lentiviral vector containing the correct ARSA gene and then reintroduced into patients. In a more than 18-month follow-up, the disease did not manifest or progress in the three patients, which had a much better motor and cognitive function compared to their untreated siblings [29]. These results were the basis for a phase I/II clinical trial using the same strategy in 20 MLD patients, which were followed for 3 years post-intervention. The preliminary results of this study revealed that this ex vivo gene therapy strategy was safe and with a clear therapeutic effect [30]. An in vivo gene therapy strategy was also tested in a phase I/II clinical trial, CERE-120 OXB-102 AAV2-GDNF AAV2 Injection in the putamen Injection in the striatum Lentivirus TH (Tyrosine hydroxylase), AADC (Aromatic amino acid dopa decarboxylase), and GCH1 (GTP cyclohydrolase I) NRTN, (Neurturin) Lentivirus TH (Tyrosine hydroxylase), AADC (Aromatic amino acid dopa decarboxylase) and GCH1 (GTP cyclohydrolase I) GDNF (Glial cell line-derived neurotrophic factor) AAV2 Injection in the striatum and the putamen Injection in the putamen AAV2 Injection in the subthalamic nucleus Injection in the subthalamic nucleus Injection in the putamen Delivery route Injection in the striatum AADC (Aromatic L-amino acid decarboxylase) AAV2 GAD (Glutamic acid decarboxylase) AAV-­ hAADC-­2 ProSavin AAV2 GAD (Glutamic acid decarboxylase) AAV-GAD Vector AAV2 Gene AADC (Aromatic L-amino acid decarboxylase) Gene therapy Name VY-AADC01 Table 9.7 Selected gene therapy clinical trials targeting Parkinson’s disease. I I/II I I/II I/II I II NCT00252850 NCT03720418 NCT01621581 NCT00627588 NCT02418598 NCT00195143 NCT00643890 Clinical trial Phase Trial code I NCT01973543 Completed Active, not recruiting Recruiting Completed Recruiting Completed Current status Active, not recruiting Terminated 9.5 Gene Therapy for Neurodegenerative Diseases 181 9 182 based on the intracerebral AAV10-mediated delivery of the correct ARSA gene in 12 different sites in 5 patients with presymptomatic or early state confirmed MLD (NCT01801709). This study was based on extensive preclinical studies, including in nonhuman primates, which showed a safe and efficient gene transfer. However, until now, no results from the clinical trials were published. Mucopolysaccharidosis type IIIA (MPSIIIA), also known as Sanfilippo syndrome type A, is caused by a recessive mutation in the SGSH (N-sulfoglucosamine sulfohydrolase) gene. It has a very early onset, around 2 years of age, with a progressive decline of cognitive and motor functions in the first decade of life, being fatal in the second decade. A first gene therapy study was launched by Lysogene, based on the intracerebral administration of AAV10 encoding both SGSH and SUMF1 (sulfatase-modifying factor) genes. The therapy was performed in four children, and the vector was injected into six different brain areas. The results showed no adverse effects and revealed some improvements or the stabilization of selected clinical parameters in some patients [31]. The company is now recruiting patients for a phase II/III clinical trial (NCT03612869), based on the administration of AAV10 vectors containing the SGSH gene (LYS-­ SAF302). Another company, Abeona Therapeutics, initiated a phase I/II clinical trial (NCT02716246) based on the systemic administration of AAV9 encoding for the normal SGSH gene. No results were yet published, although the company states in its website that the therapy is well tolerated and that positive neurocognitive signals were detected. 9.5.7 Amyotrophic Lateral Sclerosis Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is an incurable and progressive neurodegenerative disease with adult onset and a relatively short course, causing death within 3 to 5 years after the diagnosis. Neuropathologically, it is characterized by the degeneration of motor neurons in the spinal cord, brain stem and motor cortex. Gene Therapy Applications The disease includes a more common, sporadic form (affecting 90% of ALS patients), and a familial form, resulting from genetic mutations and affecting 10% of ALS patients. However, even for the familial cases, the genetic causes are complex, as ALS can arise from several mutations in at least nine different genes [32]. Maybe because of this, gene therapy studies are not abundant, with the few exceptions focused on using ASOs targeting the SOD1 (superoxide dismutase 1) gene, which accounts for 12% of the familial cases, or the C9ORF72 gene, that accounts for 40% of familial ALS cases. However, at least for the ASOs targeting the SOD1 gene, the efficacy results were not as promising as expected. For sporadic ALS forms, the development of gene therapy strategies is even more complex and difficult, as the causing factors are not clearly elucidated. Several preclinical studies in animal models showed important improvements in neuropathological and motor deficits upon viral delivery of trophic factors- coding genes, such as GDNF (glial cell line-derived neurotrophic factor), IGF-1 (insulin-like growth factor 1) and VEGF (vascular endothelial growth factor). However, despite the positive results obtained, none of these strategies has yet advanced to clinical trials. 9.5.8 Polyglutamine Diseases Polyglutamine diseases constitute the largest group of inherited monogenic neurodegenerative diseases. For this reason, and despite being rare, the nine polyglutamine diseases occupy a central stage in the development of gene therapy strategies. Several examples of preclinical and clinical studies were mentioned in Chaps. 6 and 7, especially for Huntington’s disease and Machado-­Joseph disease (also known as spinocerebellar ataxia type 3). Moreover, different approaches based both on cell and gene therapies were recently reviewed [33], highlighting the existence of extensive results from preclinical studies that support some ongoing clinical trials and certainly several others that will be developed in the future.