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.