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10 Fig. 1.4 Overview of the delivery systems used in gene therapy. Organisms and cells have developed several barriers to prevent the entry of exogenous genetic material. Therefore, overcoming these barriers to deliver the therapeutic gene is crucial to the success of gene therapy. In a broad manne...

10 Fig. 1.4 Overview of the delivery systems used in gene therapy. Organisms and cells have developed several barriers to prevent the entry of exogenous genetic material. Therefore, overcoming these barriers to deliver the therapeutic gene is crucial to the success of gene therapy. In a broad manner, delivery systems for gene therapy can be 1 Gene and Cell Therapy classified into two groups: non-viral vectors and viral vectors. The first group refers to physical and chemical methods, such as microinjection or cationic liposomes. On the other hand, the second group is based on engineered recombinant viruses that are used to deliver the therapeutic transgene. 1.8 Cell Targeting 11 Table 1.4 Main advantages and disadvantages of non-­ viral and viral systems to used deliver therapeutic genes Advantages Non-viral systems Easy production Low toxicity Unlimited cloning capacity Viral systems High efficiency in gene transfer both in vivo and ex vivo Persistence of therapeutic strategy (in some cases) High variety of cells able to be transduced High variety of viruses to be engineered Natural tropism to infect/transduce cells Natural DNA transport mechanism into nucleus (in some cases) Disadvantages Low efficiency/low expression No transgene integration Low tropism Possible immune/ inflammatory response Safety/toxicity concerns Limited cloning capacity Complex production Limited tropism (in some cases) Possibility of mutagenesis Limited knowledge on molecular mechanisms of infection ence of tetracycline alters the conformation of the TetR domain of tTA, which prevents the binding to tetO sites and blocks the transgene expression (Fig. 1.5, upper panel). From a gene therapy ­clinical view, the Tet-off system is not very suitable, as long-term administration of tetracycline might be needed in order to repress transgene expression. To overcome this problem, the Tet-on system was developed, allowing the activation of transgene expression in response to the presence of tetracycline. This was achieved by a mutation in the TetR, which allows it to function in a reverse way (rTetR), binding to tetO in the presence of the effector. The system is completed by the fusion of AD to reverse-tTA (rtTA) that binds Ptet and activates the transcription of the downstream transgene in the presence of tetracycline (Fig. 1.5, lower panel). Several improvements on these and other systems were made and are already used in gene therapy clinical trials, namely in cancer [22]. 1.8 Cell Targeting In most human diseases, different cells and organs are not affected, and thus a gene or cell therapy must ensure preferential treatment of the affected cells and organs. Targeting specificity will increase the therapy efficacy, raising the concentration of the therapeutic molecule in the most affected cells/organs and avoiding its sequestration, dilution, or inactivation in non-­target cells, at the same contributing for the increase of the safety profile of the therapy. Taking into account the existing experience with conventional drugs, as well as with cellular transplants, several strategies could be employed in gene therapy aiming at specific targeting [23]: (i) physical strategies, where the molecule/cell is delivered locally into the target area; (ii) physiological strategies, based on natural physiological mechanisms of distribution; and (iii) biological strategies, based on biological alterations of the vehicles to achieve a specific localization (Fig. 1.6). Local delivery is probably the most straightforward way of administering a gene therapy; nevertheless, the procedure is extremely invasive, and particular cells/organs may be difficult to access. Taking advantage of physiological mechanisms such as blood circulation is another strategy for gene delivery. Despite the fact that it could be used in some contexts, systemic delivery has to deal with several physiological barriers, for example the BBB, when accessing the central nervous system. The biological strategy implies the modification of the vector/cell, altering its entry proprieties or modulating post-entry features, for example by using a promoter specific to the target cells (Table 1.5) [24]. Depending on the type of vector (viral or non-viral), different modifications could be made in the surface of the vector (mode details on these modifications are detailed in Chaps. 2 and 3). As an example, the lentiviral vector envelope can be modified using glycoproteins from other viruses, thus altering its tropism. In the case of adeno-­associated virus (AAV), different serotypes have tropism to different cells, providing a wide 1 12 Gene and Cell Therapy Fig. 1.5 Tetracycline (Tet) regulation system for the control of gene expression. This system is based on the bacterial Tet operon and the administration of tetracyclines or their derivates to control gene expression. The Tet system has two main variants: the Tet-off system (upper panel) and the Tet-on system (lower panel). In the Tet-off system, the TetR is modified with a transcription activation domain from the VP16 protein of the herpes simplex virus, creating a tetracycline-controlled transcriptional activator (tTA). Additionally, the tetO sequences are fused with a TATA box-containing eukaryotic promoter to construct the tetracycline-responsive promoter (Ptet). In the absence of tetracycline (or its derivates), the tTA will bind to the tetO sites in the Ptet, thus activating the expression of the downstream transgene. On the other hand, the presence of tetracycline alters the conformation of the TetR domain of tTA, which prevents the binding to tetO sites and blocks the transgene expression. On the contrary, the Tet-on version allows the activation of transgene expression in the presence of tetracycline. This is achieved by a mutation in the TetR, which allows it to function in a reverse way (rTetR), binding to tetO in the presence of the effector. The system is completed by the fusion of AD to reverse-tTA (rtTA) that binds Ptet and activates the transcription of the downstream transgene in the presence of tetracycline. range of selectable choices. Other viruses like herpes simplex virus have a natural tropism to neurons, which makes them particularly suitable as delivery systems to the nervous system. In the case of non-viral vectors, especially the chemical-based vectors, the use of additional molecules helps to ensure a more directed targeting of particular cells. For example, the use of transferrin allows liposomes to enter the brain, surpassing the BBB. 1.9 Immune Response to the Therapy Circumventing the immune response is a major issue in gene therapy (except if the goal is vaccination or tumor lysis), especially when using viral vectors (see Chap. 4 for more details). The immune system comprehends a complex array of mechanisms protecting the body against 1.9 Immune Response to the Therapy Fig. 1.6 Cell targeting strategies in gene therapy. The delivery of the therapeutic gene to the target cells/organs can be performed using physical strategies, in which there is a local and direct delivery of the gene using devices, such as catheters, or by using physiological strategies, in which endogenous physiological mechanisms, e.g., the blood circulation, are used to deliver the therapeutic gene. Finally, different biological strategies that take advantage of different biological mechanisms can also be used to tar- 13 get cells. For example, the therapeutic gene and vector can have a natural tropism to the target cell or organ, while not targeting other cells. Another strategy involves the use of specific molecules to be recognized by specific target cell receptors, thus specifically delivering the therapeutic gene to these cells. Finally, the use of cell-specific promoters could also limit the expression of the therapeutic gene to the target cells. 1 14 Table 1.5 Different gene promoters and their cell specificity Promoter CMV PGK UbC hAAT TBG Desmin MCK Synapsin CaMKII mGluR2 GFAP MBP Cytomegalovirus Phosphoglycerate kinase Ubiquitin Human α-1-antitrypsin Thyroxine-binding globulin Desmin Muscle creatine kinase Synapsin Ca2+/calmodulin-­ dependent protein kinase II Metabotropic glutamate receptor 2 Glial fibrillary acidic protein Myelin basic protein Specificity Ubiquitous Liver Skeletal muscle Neurons Astrocytes Oligodendrocytes Gene and Cell Therapy Several strategies were developed to ensure the success of gene therapy by overcoming the immune response [26]. One strategy is to avoid the expression of the delivered gene in antigenpresenting cells (APCs), such as dendritic cells, B-cells, or macrophages. The regulated expression of the transgene could also be used as a strategy to avoid the immune response, by delaying the expression of the gene until the tissue recovers from the inflammation associated with the vector administration. Another strategy is to deliver the genes into immune-privileged sites, like the brain or the eye. Additional strategies also include the modifications of the vector used, for example, performing genetic (viral vectors) or chemical and nonchemical modifications to its structure (both viral and non-viral vectors). Finally, another type of strategy is based on immunosuppression, similar to the procedure following organ transplantation. However, the use of immunosuppression strategies should be carefully planned, as they could interfere with other aspects of the gene therapy, such as modifying the vector internalization, stability, and transduction efficiency or leading to long-term complications such as the increased risk of malignancies. p­ athogens like viruses and bacteria. The system is divided into two main responses: (i) the innate immune response, which is the initial, rapid and unspecific defense, and (ii) the adaptive immune response, which is stimulated later and more complex than the innate response. The latter response involves the specific, antigen-­1.10 Highlights in the History mediated, recognition of the pathogen, its elimiof Gene and Cell Therapy nation through humoral and/or cellular responses, and a memory component that allows improved Many events contributed to gene therapy develresistance to future infections. opment; thus, it is not easy to select the main Both viral and non-viral vectors can induce an highlights, taking also into account that for many immune response, which could lead to the elimi- of these important marks to happen, a lot of studnation of the vector and of the transduced cells, ies and developments were also made. Several of thus decreasing the efficacy of the therapy. the important milestones that we selected for Moreover, the production of proinflammatory these highlights were already referred, including cytokines and chemokines also has a harmful the first gene therapy clinical trial or the first effect on the organism. Diverse factors influence approved gene therapy product in Europe. It is the immune response to a vector, including [25] also important to note that several discoveries (i) the route of administration, (ii) the dose of the and studies were not related directly to gene thervector, (iii) patient-related factors (e.g., age, gen- apy, but contributed decisively for its developder, immune status, drug intake), (iv) the type of ment, taking as an example the discovery of the promoters and/or enhancers used, and (v) the RNAi mechanism. Furthermore, several advances alterations made to the vector genome sequence related to cell therapy were also important for the and/or structure. gene therapy history, for example, the development of iPSC. 1.10 Highlights in the History of Gene and Cell Therapy It is virtually impossible to date the beginning of gene therapy. However, two pioneer papers are probably in the genesis of the idea and possible implications of human gene therapy. In 1967, Marshall Nirenberg formulated the question: Will society be prepared?, recognizing that genetic messages could be synthesized chemically and then be used to program cells [27]. The author recognized the enormous potential of this approach but also the obstacles and ethical problems behind it. Later, in 1972 Friedmann and Roblin asked: Gene therapy for human genetic disease? [3]. If these two papers probably gave the starting shoot on the discussion about gene therapy and its implications, two important scientific advances contributed decisively for the concretization of gene therapy: the first virusmediated gene transfer in 1968 [28] and the creation of the first recombinant DNA molecule in 1972 [29]. As pinpointed throughout this book, the delivery system of the gene is essential to the success of gene therapy and, since the first attempts, a lot of effort was directed to that development. Therefore, another important advance for gene therapy came with the construction of a retrovirus vector. In 1984, Cepko, Roberts, and Mulligan [30] reported the development of a murine retrovirus vector, which allowed the efficient introduction of DNA into mammalian cells. The first approved protocol to introduce an exogenous gene into humans was approved in 1988 and the study published in 1990 [31]. The study was not therapeutic, but rather described the introduction of a bacterial gene into tumor-­infiltrating lymphocytes and the tracking of the persistence and localization of the cells after reinfusion into patients with advanced melanomas. Also in 1990, the first gene therapy clinical trial for ADA-SCID took place [4] and launched gene therapy interventions in humans. The initial idea was to perform ex vivo gene therapy using autologous hematopoietic stem cells (HSCs) and retrovirus; however the preliminary studies in nonhuman primates were disappointing, with low levels of viral transduction and engraftment. Instead, the researchers used autologous T-cells 15 treated with the functional ADA gene delivered by a gamma retrovirus vector, which were stimulated to divide in vitro and then the cells were reinfused into the patients. More than the success of the intervention, this clinical trial was a mark for gene therapy, as it proved that it could be applied in humans in a feasible and safe manner. Until now, dozens of ADA-SCID patients have been treated using gene therapy with enormous success, opening the way for the approval of Strimvelis® to treat ADA-SCID, which was the second gene therapy product approved in Europe. In 1998, an important discovery that contributed decisively to the success of gene therapy was published. Scientists Andrew Fire and Craig Mello discovered RNAi [15], a mechanism of gene expression regulation, based on small RNA molecules (siRNAs and microRNAs) complementary to messenger RNAs (mRNAs), which activates a degradation pathway for those mRNAs. But what is the importance of RNAi to gene therapy? In the classical view of gene therapy, a functional or normal copy of a malfunctioning gene is introduced in a patient to treat a disease. Theoretically, in some recessive conditions, this intervention will lead to a complete cure, as one single functional copy may be enough to prevent the disease phenotype. However, for dominant conditions, the introduction of a normal gene is not enough to revert the disease. In this sense, RNAi offered the possibility to treat dominant diseases with gene therapy, by using these small RNA molecules. These molecules can be designed to be complementary to the target mRNA, leading to a reduction of target protein levels and in theory to the mitigation or even cure of the disease phenotype. The use of RNAi or antisense oligonucleotides greatly contributed to the development of gene therapy silencing strategies, aiming to abrogate or reduce the expression of a defective protein causing a disease. In 1999, an important complication that negatively impacted the development of the gene therapy field occurred, with the death of Jesse Gelsinger in a clinical trial for ornithine transcarbamylase (OTC) deficiency. This is a metabolic 16 disease that affects ammonia elimination, being fatal in the first days after birth. However, some patients have a partial OTC deficiency that may be controlled by a strict diet and pharmacological drugs. Jesse Gelsinger had this partial deficiency and was considered an ideal candidate for the gene therapy intervention. He received a dose of 3.8 × 1013 recombinant adenoviral vectors containing the normal OTC gene directly in vivo in the hepatic artery [7]. Jesse died 4 days after the intervention, due to a severe immune reaction to the vector, which induced shock syndrome, cytokine release, acute respiratory distress, and multi-­ organ failure. It is, however, important to refer that the other 17 subjects that participated in this study, including asymptomatic ones, presented transient mild adverse effects such as muscle aches and fevers. It is also very important to point out that by February 2000 (some months after Jesse death), more than 4000 subjects were already subjected to gene therapy in approximately 400 clinical trials, and Jesse was the only reported death [32]. Nevertheless, at that time, several clinical trials of gene therapy were halted, reviewed, or suspended, and, in the USA, FDA and the National Institutes of Health (NIH) promoted the development of new two programs, trying to enhance the monitoring of gene therapy clinical studies. In 2000, a gene therapy clinical trial conducted in Europe was published in Science, reporting the treatment of 10 boys with a type of immunodeficiency (X-SCID) [8]. The treatment was successful, and 10 years after the trial, the disease was corrected [9]. However, the insertion site of the therapeutic gene led to the development of leukemia in four of the treated boys, and one died because of that. Following this event, FDA suspended gene therapy clinical trials in 2003. Contrastingly, in 2003 China approved the first gene therapy product, Gendicine®, which started to be commercialized in 2004. The product consists of the p53 gene delivered in an adenoviral vector aiming to treat patients with head and neck squamous cell carcinoma [11]. The reported data showed very good therapeutic results and no major adverse side effects, and until 2013 more than 10,000 patients were treated 1 Gene and Cell Therapy with Gendicine® for different types of cancer [33]. Despite all of this, the product was never approved in Europe, the USA, or Japan. The halt in the clinical trials did not stop the development and improvement of gene therapies and strategies. In 2004, EMA granted the first commercial Good Manufacturing Practice (GMP) certification in the EU for the production of commercial supplies of gene-based medicines. The product licensed was Cerepro®, which is an adenoviral vector with the gene thymidine kinase from herpes simplex virus aiming to treat malignant brain tumors. Despite several clinical trials, including a phase III trial, the product did not receive marketing authorization from EMA. In 2004, an important advance for science in general and for gene therapy, in particular, was achieved. In a major international effort, the human genome was fully sequenced, thus completing the Human Genome Project [34] and providing the possibility of precisely locating all human genes. Among other important applications, human genome mapping provided a framework for the development of gene editing techniques for gene therapy. In 2006, another major breakthrough was published by Shinya Yamanaka, which reported the development of induced pluripotent stem cells (iPSC) [35]. These pluripotent stem cells were reprogrammed from adult fibroblasts using four genes, now known as the Yamanaka reprogramming factors: Oct3/4, Sox2, Klf4, and c-Myc. The potential of this advance was soon perceived, namely for regenerative medicine. In the field of gene therapy, it expanded the possibilities of using autologous cells in ex vivo gene therapy applications. In 2014, the first application of iPSC-derived cells in humans started in Japan, for macular degeneration, which is the most prevalent retinal disease in aged people [36]. Despite the reported positive results, the clinical trial was halted 1 year later, due to safety concerns [37]. In the following years, constant research aiming at improving the safety of delivery vectors and at developing better assays for risk assessment led to the return of gene therapy clinical trials for different conditions, such as Leber’s congentinal amaurosis, β-thalassemia, or hemo- 1.12 Ethical Questions and Concerns About Gene and Cell Therapy philia B [38]. The continuous push and effort by many researchers both in preclinical and clinical gene therapy studies led to the approval [39] in 2012 of the first gene therapy product in Europe, Glybera®. The product was based on the delivery of a functional copy of the lipoprotein lipase gene by an AAV vector. However, at the end of 2017, the company producing the product did not renew the marketing authorization, and it was removed from commercialization. More recently, the development of techniques based on nucleases to precisely edit the genome brought a new advance to the gene therapy field, with the possibility of treating the genetic cause of a disease by directly editing or replacing the causative gene(s). These technologies utilize meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced palindromic repeats (CRISPR) systems. ZFN was the first gene editing system reaching a phase I clinical trial [40]; however, currently most of the studies and research is directed to the CRISPR-Cas system. The very high efficiency and an easy and rapid construction provided an enormous boost in its use compared to the other gene editing plastforms. In 2016, the first clinical trial using the system was approved and launched in China [17]. Gene therapy has come a long way, following a path of successes and drawbacks since the first clinical trial back in 1990. A huge effort by scientists, clinicians, and biotech companies led to the development and continuous improvement of the techniques, systems, and safety of gene therapy applications. The future is of course unknown; nevertheless the recent approval of several gene and cell therapy products in Europe and the USA might indicate that gene therapy is here to stay. 1.11  urrent Status of Gene C Therapy Until November 2017, more than 2600 approved gene therapy clinical trials took place around the world (Fig. 1.7) [41]. Most of these clinical trials were directed to treat cancer, followed by mono- 17 genic, infectious, and cardiovascular diseases (Fig. 1.8). The recent boost in gene therapy and its related safety and technical developments led to the approval of 13 gene therapy products in Europe and/or the USA (Table 1.6). Several others are in the final stages of development and could receive marketing authorization in the near future. 1.12 Ethical Questions and Concerns About Gene and Cell Therapy Considering several issues already mentioned in this book, it is clear that gene therapy is very prone to raise important ethical questions, controversies, and debates. It is evident that genetic manipulation comes with many disadvantages, but also with enormous potential and opportunities (Table 1.7). Thus, gene therapy, as any other medical intervention involving human subjects, has to important requirements and considerations, such as (1) guaranteeing informed consent, considering the ethical principle of respect for persons; (2) having a favorable risk-benefit balance, considering the ethical principle of beneficence/non-maleficence; and (3) having a fair and rigorous selection of the research subjects, considering the ethical principle of justice. However, the complex issues surrounding gene therapy, such as the possibility of altering the personal genetic information, raise specific ethical quandaries: Should germline gene therapy be allowed? How to distinguish between gene enhancement and gene therapy? How to regulate the application of gene therapy? Do we understand the full implications of gene alterations? Is gene therapy only available to people with higher monetary incomes? And so on. A fundamental ethical question in gene therapy concerns the somatic versus germline gene therapy debate. From the accepted point of view, all gene therapies in human subjects should target somatic cells; however, even in this case, enhancement and safety problems should be contemplated. The prohibition on gene therapy targeting the germline cells ensures that its 18 1 Gene and Cell Therapy Fig. 1.7 Gene therapy clinical trials approved by the end of 2017. More than 2500 clinical trials using gene therapy were performed worldwide until 2017. Fig. 1.8 Human conditions addressed in gene therapy clinical trials. Different gene therapy strategies have already been applied to different human conditions, espe- cially to cancer (65% of all the gene therapy clinical studies performed), monogenic diseases (11.1%) and cardiovascular diseases (6.9%). nonethical application is prevented altogether. This issue is now even more relevent, with the development of gene editing techniques allowing the direct and locally specific modification of genes in cells and patients, which fomented novel ethical questions and concerns around gene therapy. Even though the matter of not performing germline gene therapy in humans was a relatively consensual issue among the medical and scientific communities, the recent claim of human embryo editing sounded the alarm and reinforced the regulatory concerns. Although the gene editing procedure was not yet completely verified and certified, the very possibility that is true united the scientific and medical communities against that type of manipulation. Something that undoubtedly emerges from these controversies and debates is the need for a clear regulation on gene therapy application in human subjects, which could be seconded by most of the developed and developing countries having the technology and the means to apply it. Kymriah Luxturna Yescarta Strimvelis Zalmoxis Kynamro Spinraza Exondys 51 Onpattro (patisiran) Tegsedi (inotersen) Zolgensma Zynteglo 1 2 3 4 5 6 7 8 9 10 13 Ex vivo In vivo In vivo In vivo In vivo In vivo In vivo Ex vivo Ex vivo Ex vivo In vivo Ex vivo Administration route In vivo BA-T78-Qglobin SMN1 ASO ASO ASO RNAi ΔLNGFR and HSV-TK Mut 2 ASO CD19-­specific CAR T ADA CD19-­specific CAR T RPE65 Gene GM-CSF CAR T chimeric antigen receptor T-cell a Caused by mutations in both copies of the RPE65 gene b Per eye c Per infusion d The first infusion and then 375,000 per year for life e Per year f Currently under review by EMA 12 11 Product Imlygic Lentiviral vector AAV9 – – Lipid nanoparticles – – Lentiviral vector Adeno-­ associated vector Y-Retroviral vector Retroviral vector Retroviral vector Vector HSV-1 β-thalassemia Homozygous familial hypercholesterolemia (HoFH) Spinal muscular atrophy (SMA) Duchenne muscular dystrophy Hereditary transthyretin-mediated amyloidosis Hereditary transthyretin-mediated amyloidosis Spinal muscular atrophy (SMA) Received a stem cell transplant ADA-­SCID Non-­Hodgkin lymphoma (NHL) Acute lymphoblastic leukemia (ALL) Inherited retinal diseasea Indication Melanoma Table 1.6 Current gene therapies approved for human use in Europe and the USA. Intravenous infusion Cell transplant Intrathecal injection Intravenous Intravenous infusion Subcutaneous Injection Cell transplant Cell transplant Yes Nof Yes Yes No Yes No Yes Yes No No Subretinal injection Cell transplant Yes Approval in Europe Yes Targeting Intralesional injection Cell transplant No Yes Yes Yes Yes Yes Yes No No Yes Yes Yes $2.1 million €1,600,000 $450,000e $750,000d $300,000 $450,000e $176,000 €149,000c €594,000 $373,000 $425,000b $475,000 Approval in the USA Price Yes $65,000 1.12 Ethical Questions and Concerns About Gene and Cell Therapy 19 1 20 Table 1.7 Main advantages and disadvantages of gene manipulation Advantages Less risk of genetic diseases Possibility of preventing disease transmission to next generations Increase in life expectancy Increase in the quality of life Disadvantages Access could be limited to high income individuals Reduction of genetic diversity Unknown interaction between genes Irreversiblte alteration of the human gene pool Gene and Cell Therapy design and implementation. • The story of gene therapy is full of advances and setbacks; however, there are currently several gene therapy products approved in Europe and the USA. • Several ethical problems arise with gene therapy, as it became evident in 2018, with the claim of the first human embryo gene editing procedure with the CRISPR-Cas system. Review Questions However, the huge advance in technical aspects and their availability to almost everyone will certainly constitute an important and difficult challenge for authorities and for the scientific community. This Chapter in a Nutshell • Gene therapy encompasses a set of strategies for modifying gene expression or correcting mutant/defective genes, involving the administration of nucleic acids. • Somatic gene therapy refers to the interventions targeting somatic cells. • Germline gene therapy targets the reproductive cells, which could affect the progeny genetic information. • There are several types of gene therapy: (a) gene augmentation, (b) gene silencing, (c) gene editing, and (d) suicide gene therapy. • The direct administration of gene therapy is called in vivo therapy, whereas the treatment involving gene therapy in cells and then the transplant to the organism is called ex vivo therapy. • Two main delivery systems exist, the viral and the non-viral vectors. The former systems have a high efficiency compared to the non-­viral systems, although their safety profile is lower. • There are several strategies to ensure the gene therapy expression, persistence, and targeting, for example using gene regulation systems. • The immune response to gene therapy is also an important consideration in clinical trial 1. Which sentence better defines gene therapy? (a) A method to cure genetic disorders (b) A method to deliver a gene (c) A method to correct a defective gene (d) A method to silence the expression of a defective gene (e) All the previous options 2. Which of the following points is not essential in the development of a gene therapy clinical trial? (a) Delivery system (b) Target cell/organ (c) Immune response (d) Civil status (e) Therapy cost 3. To have a successful gene therapy, the healthy gene should be inserted in the target cell and: (a) Destroy the defective gene (b) Produce the correct amount of normal protein (c) Bind to mRNA molecules in the cell (d) Be inserted in the mitochondria (e) None of the previous 4. From the following, which is a disadvantage of gene therapy? (a) Eradicate diseases (b) Prevent diseases (c) High cost (d) Enormous potential (e) Correct genetic defects 5. The first clinical trial and the first death caused by gene therapy had as a disease target (choose the correct answers): (a) Adenosine deaminase deficiency