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Soleimani, Merheb, and Matar: Human gene therapy – the future of health care


‘I am sorry. There is nothing more we can do.’ That is what a doctor would say to a patient or the parents of a fetus suffering from, what might seem at this point, an incurable genetic disorder. The string of similar clichéd apologies was broken for the first time in 1990 by two brave souls, Dr Anderson and Dr Blaese, who took the initiative by treating a 4-year-old girl lacking an adenosine deaminase (ADA) gene with a role in immune responses.1 Gene therapy is a technique to alter aberrant phenotypic characteristics caused by mutations through approaches such as gene insertion, modification or regulation.2 The technique is particularly advantageous over conventional medications in that it aims to correct the underlying cause of the disease, rather than its symptoms. Cancer, cardiovascular diseases and multiple sclerosis are some of the conditions with prospects for treatment with gene therapy. In addition, there have been attempts to tackle inherent genetic disorders at the embryonic stage. This form of gene therapy, however, has given rise to enormous controversy arising from the fear of misusing the technique for non-medical purposes, uncertainties regarding its success and other ethical issues. To address the issues, stringent regulations may be necessary; however, standing in the way of gene therapy merely out of fear is deemed unreasonable.

Historical perspectives

The idea of manipulating the genome emerged in the 1960s as the antisense concept, following the discovery of the DNA double helix and the explanation of its replication, expression and regulation.3 Joshua Lederberg was one of the pioneers who envisaged the possibility of controlling nucleotide sequences in human chromosomes and grafting DNA segments onto viral DNA.4 The hypothesis was reinforced during the 1970s, mainly as a result of the discovery of reverse transcriptase and the comprehensive studies on restriction enzymes.3 Soon after came two unapproved clinical trials: one to treat the arginase deficiency syndrome using the Shope papilloma virus, and the other to counteract β-thalassemia with bone marrow cells treated with a β-globin-containing plasmid.5 Despite the success of treating ADA in 1990, the decade that followed was full of disappointments, the biggest of which occurred in 1999 with the death of a teenager who had undergone gene therapy for a rare liver disease.6 With improvements in our understanding of vectors, gene therapy has taken a sharp turn for the better, promising (science-grounded) miracles in the future. A concise timeline of the most salient events that have shaped gene therapy is shown in Table 1.


Timeline of the most important events in the history of gene therapy

1959 Physician Stanfield Rogers observed that the warts caused by the Shope papilloma virus on the skin of rabbits were rich in arginase, which he attributed to the introduction of a Shope virus-encoded arginase gene into rabbit skin cells3
1968 Nobelist Marshall Nirenberg discussed programming cells with synthetic messages7
1970–1973 Stanfield Rogers used the Shope papilloma virus in an unsuccessful attempt to treat two sisters suffering from hyperargininaemia, as he believed the virus would induce arginase activity in the patients8
1974 Creation of the Recombinant DNA Advisory Committee (RAC) to stringently regulate all gene transfer experiments (
1980 Martin Cline conducted an unapproved experiment to transfer the β-thalassemia gene into the bone marrow of two patients with hereditary blood disorders9
1983 Miller et al.10 built a retroviral vector and used it to transfer a human hypoxanthine-guanine phosphoribosyltransferase gene into rodent cells in vitro10
1984 The RAC created the Human Gene Therapy Working Group specifically to review gene therapy protocols8
1990 Anderson and Blaese cured a young girl suffering from severe combined immunodeficiency by injecting her with T cells carrying an adenosine deaminase gene11
1997 A HIV-positive child was treated using recombinant CD34+ cells carrying a HIV-1 PRE decoy gene12

HIV, human immunodeficiency virus; PRE, progesterone-responsive element.

Gene therapy: approaches, types and vectors

In brief, gene-based therapeutics (gene therapy) may be generally defined as altering the expression of specific genes or correcting defective genes by introducing exogenous nucleic acids in order to prevent, arrest or reverse a pathological process.13 There are three chief approaches that are adopted to carry out gene therapy. Gene addition/insertion is the most common approach and involves introducing into cells a normal copy of a non-functional, mutated gene that is otherwise responsible for the production of a key protein. This method has been shown to tackle genetic disorders such as haemophilia14 and cystic fibrosis,15 which are characterized by the lack of the clotting factor IX and a chloride channel, respectively. The genes encoding these proteins are delivered into cells where they are expressed to provide the patient with the protein that they lack. Gene knockdown is the second approach, which entails regulating and/or inhibiting the expression of a mutant gene with the help of double-stranded RNA (dsRNA) via the process of RNA interference (RNAi). RNAi is a biological process whereby endogenously produced or artificially introduced dsRNA binds to the target messenger RNA (mRNA) and causes its degradation and, therefore, silences the target gene.13,16 Being a biological response, RNAi can be triggered in patients by the administration of short synthetic dsRNAs or by the expression of short hairpin RNAs (shRNAs). Its mechanism involves a series of events during which dsRNAs and shRNAs are processed mainly by an enzyme called Dicer to generate short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. siRNAs bring about cleavage of their target mRNAs and can, therefore, be used to completely suppress disease-causing genes. miRNAs, on the other hand, follow a non-cleavage-dependent pathway, which renders them useful in modulating gene expression.17 The therapeutic applications of RNAi include, but are not limited to, the treatment of cancer,1820 Alzheimer’s disease,21 rheumatic diseases22 and eye diseases.23,24 Gene correction/modification is another approach wherein zinc-finger nucleases and DNA recombination techniques are applied to either repair a mutation or mutate a gene, encoding a virus receptor in order to thwart infection.25 Zinc-finger nucleases are powerful gene modification tools composed of a sequence-specific, customizable DNA-binding domain and a nuclease domain that functions only when bound to the target site.26 The cleavage of DNA evokes a damage response that is prominently in the form of non-homologous end joining which, despite being inaccurate, can be used to introduce small insertions and/or deletions to inactivate mutant genes.25,27 To augment the accuracy of the response, a synthetic donor DNA template homologous to the target is delivered along with zinc-finger nucleases to replace the target with desired genetic information through homologous recombination.27,28 The method has been utilized in the treatment of HIV29 and diseases such as haemophilia,30 sickle-cell anaemia31 and the X-linked chronic granulomatous disease,32 and also in the creation of knockout models.33 Alternatively, a donor template that has only one mismatch with the target gene can be used to induce a site-specific correction of a point mutation in a process referred to as site-directed mutagenesis. A more commonly practised approach involves the use of RNA/DNA oligonucleotides, as they are more active in homologous pairing than DNA alone.34 Alexeev et al.35 used this approach to demonstrate that injection of albino mice with RNA/DNA oligonucleotides tailored to correct a point mutation in the tyrosinase gene would result in permanent and inheritable restoration of tyrosinase activity and melanin synthesis. Other disorders against which this method has been employed include sickle cell anaemia,36 Fabry’s disease34 and haemophilia B.37

Depending on the type of target cell, gene therapy is broadly classified as somatic or germline. Clearly, the former refers to correcting anomalies in non-reproductive cells in the patient, whereas the latter is often performed in early embryos, such that the change will be passed on to posterity.38 Somatic gene therapy could be implemented either in vitro or in vivo. The in vitro method consists of three key steps: (1) transforming a virus and suppressing its ability to reproduce; (2) transfecting the virus into cells isolated from a patient; and (3) injecting the genetically modified cells into the patient.39 Conversely, the in vivo method involves direct administration of injectable self-eliminating vectors (e.g. adenoviral vectors) to patients.40 Germline gene therapy is relatively cumbersome and sensitive with a protocol that comprises eight major steps: (1) isolation of undifferentiated cells from an embryo or a gamete; (2) culturing the cells in vitro; (3) transfection of the cells; (4) selection of the transfected cells; (5) replacement of the targeted gene; (6) removal of marker genes; (7) nuclear transfer into an unfertilized ovum; and (8) reimplantation of the ovum in the uterus.38

Vectors play a crucial role in transferring DNA into cells. They fall under the two classes of viral and non-viral vectors. Viral vectors are essentially viral particles composed of nucleic acid contained within a capsid protein that is, more often than not, covered by a membrane envelope. Generally, some of the structural genes of these vectors are deleted in order to prevent them from replicating in host cells.41 Gene delivery by means of replication-incompetent viral vectors has been demonstrated to be more efficient and less demanding in terms of dose number because of the presence of strong promoters in viral vectors.42 The genetic material of each type of viral vector may be different from those of others, which confers unique features for the therapeutic exploitation of the virus (Table 2).


Genetic material, advantages and disadvantages of viral vectors43

Viral vector Genetic material Advantages Disadvantages
Retroviral vectors dsRNA Insert capacity for transgene < 7–8 kb
Stable integration into host DNA
Low immunogenicity
Broad cell tropism of infectivity
Prolonged expression
Difficult targeting of viral infection
Transfect only proliferating cells
Concern of insertional mutagenesis
Lentiviral vectors dsRNA Infect proliferating and non-proliferating cells
Stable gene expression
Potential insertional mutagenesis
No clinical trials
Adenovirus vectors dsDNA High transfection efficiency
Efficient targeted transfection
Infects dividing and non-dividing cells
Immune response to viral proteins
Insert size limit of 7.5 kb
Transient gene expression
Herpes virus vectors dsDNA Infects a wide variety of cell types
Insertion capacity of up to 50 kb
Generation of high virus titres
Possible toxicities
Risk of recombination
Poxvirus vectors dsRNA High insertion capacity
High expression levels
Possible cytopathic effects
Adeno-associated virus (AAV) dsDNA Infect dividing and non-dividing cells
Very prolonged expression
Low immunogenicity
Insert size limit of 4.5 kb
Insufficient clinical trials
Requirement of adenovirus or herpes virus for AAV replication

dsDNA, double-stranded DNA.

Notwithstanding their high efficiency and specific uptake, viral vectors are plagued by their stimulation of the host immune system that recognizes the vector and the transgene product(s) as a foreign agent.44 Non-viral vectors are deemed superior to viral vectors in some respects as they are safer, can be more easily constructed and modified and display a higher packaging capacity.45 Typically, non-viral vectors are in the form of either naked DNA or a complex of DNA and nanoparticles.46 Liposomes are known to be the most popular type of nanoparticles composed of natural or synthetic phospholipids and steroids or other surfactants. Thanks to their ability to fuse with biological membranes, liposomes have been successfully employed to transfect DNA into cells.47 Polymers constitute another class of non-viral vector for therapeutic gene delivery. The most studied polymers are dendrimers that are highly branched macromolecules, consisting of a central atom (e.g. nitrogen), interior layers of polymers and an exterior, cationic functionality layer.48 Microprojectiles such as tungsten and gold nanoparticles coated with DNA also serve as effective non-viral vectors that are blasted into target cells using a gene gun.46 Other notable modes of delivery for non-viral vectors, in general, include electroporation (relies on electrical pulses to create pores in the cell membrane),49 ultrasound50 and hydroporation (uses a highly pressured aqueous solution to generate membrane pores).51

Applications: case studies

As far as its applications are concerned, gene therapy is a versatile tool, potentially capable of treating a range of disorders that require gene correction or replacement. Cancer remains, by far, the most investigated potential application of gene therapy (Table 3). The most successful clinical trials, however, are those that address cardiovascular diseases.52 This review will discuss specific studies in the application of gene therapy against colon cancer, heart failure, multiple sclerosis and type 1 diabetes mellitus.


Proportions of gene therapy clinical trials for various diseases52

Disease/indication Proportion of trials (%)
Cancer 64.4
Monogenic diseases 8.7
Cardiovascular diseases 8.4
Infectious diseases 8.0
Neurological diseases 2.0
Ocular diseases 1.5
Inflammatory diseases 0.7
Other diseases 1.4
Gene marking 2.7
Healthy volunteers 2.3

Given their high death toll, cancers, both benign and malignant, are relentlessly investigated for the possibility of using gene therapy as a form of treatment. Researchers are constantly improving the technique for the purpose of presenting it as a safe and superior alternative to chemotherapy, which has the potential to cause systemic toxicity and/or, in the long run, lead to other types of cancer.53 The studies in this regard are largely focused on two strategies, namely immunotherapy and oncolytic virotherapy. Briefly, immunotherapy involves use of cells transfected with cytokine genes in an attempt to provoke a robust immune response against existing tumour(s).54 Oncolysis, on the other hand, uses viruses that replicate within tumours, destroying them in the process. As a case study on oncolysis, Yoon and collaborators55 assessed the efficacy of a herpes simplex virus (HSV) mutant hrR3, containing a β-galactosidase insert within its ribonucleotide reductase gene for cancer therapy. The virus was expected to proliferate in tumours, depending on the ribonucleotide reductase supply by cancer cells. In cell culture studies, human, as well as murine, colon carcinoma and hepatocytes were infected with hrR3, and subsequently subjected to viral cytotoxicity assays. The number of surviving cells was greater in the case of hepatocytes, indicating the effectiveness of the virus as an antitumour agent. Results of animal studies were equally favourable, as mouse models suffering from carcinoma showed tumour lysis with blue (β-galactosidase) spots in the cleared regions upon infection with hrR3.55

Similar to cancer, cardiovascular diseases are frequently cited among those that gene therapy is expected to cure. These diseases include, among others, heart failure, arrhythmia and coronary artery disease.56 Both the in vivo (percutaneous) and the in vitro approaches to gene delivery may involve non-viral and/or viral vectors.57 That said, viral vectors are given preference, as they have proven more efficient and some of them (e.g. adenoviruses) can even infect the non-dividing cells of the cardiovascular system.56 Jessup et al.58 specifically exploited adeno-associated viruses in a study on 39 patients to examine the efficacy of sarcoplasmic reticulum-calcium (Ca2+)-adenosine triphosphatase (ATPase) (SERCA2a) in alleviating the symptoms of heart failure. It is worth noting that reduced calcium uptake, which has been observed in failing hearts, is closely associated with a decrease in the expression and activity of SERCA2a. To normalize intracellular calcium cycling and correct cardiac metabolism, the authors randomly administered the patients with either one of the three different doses of SERCA2a-transformed adeno-associated virus type 1 or a placebo by coronary artery infusion over a 12-month period. In the end, their results attested to the effectiveness of the method, as the patients who had received higher doses exhibited considerable improvement in functional capacity, symptoms and cardiac structure.58

Multiple sclerosis (MS) is an autoimmune disease with good prospects for a gene therapy-based cure. Patients afflicted by this chronic, neurological disease of the central nervous system experience episodes of neurological deficits in the earlier stages, followed by progressive neurological deterioration.59 Currently, a wide range of disease-modifying therapies, such as those using interferon-β (IFN-β) (down-regulating T-helper cells), glatiramer acetate (Copaxone®, Teva UK, Essex, UK; preventing lymphocyte proliferation) and mitoxantrone (inhibiting repair by topoisomerase), are in use or under study.60 However, the fact that most of these drugs do not elicit an optimal response in many patients has directed attention towards gene therapy. Ryu and colleagues61 reported the use of IFN-β as a therapeutic gene to reduce the autoimmune effects of MS. Initially, human bone marrow-derived mesenchymal cells (MSCs) were cultured and infected with adenoviral vectors transformed with either green fluorescent protein (GFP) or murine IFN-β. To induce experimental allergic encephalomyelitis (an animal model for MS), the mice were given the MOG35–55 peptide emulsified in Freund’s adjuvant containing Mycobacterium tuberculosis. Later, the models were divided into two groups, each receiving either MSCs–GFP or MSCs–IFN-β. The impact of the therapy was evaluated, taking into account its various aspects and likely consequences. Histological analysis of spinal cord sections taken from mice containing MSCs–GFP (control) and MSCs–IFN-β revealed that demyelination and infiltration were more severe in the former. In addition, the results of an enzyme-linked immunosorbent assay confirmed the immunomodulatory effect of the therapy by indicating that cytokine levels were far lower in MSCs–IFN-β recombinants. To those, one has to add the relatively high expression levels of a regeneration stimulant called the neurotrophic factor in the MSCs–IFN-β models that further added weight to the reliability of the method.61

Type I diabetes mellitus (T1DM) is another autoimmune disease, among several others, that has been shown to be a viable candidate for treatment by gene therapy. T1DM occurs as the result of the autoimmune destruction of insulin-secreting β-cells that induce the removal and metabolism of glucose by muscle and liver cells, respectively.62 T1DM is a polygenic disease, as it is caused by the combined effect of more than one underlying susceptibility gene. About 18 loci (regions) of the genome, besides environmental factors such as diet and infection(s), are known to contribute to the onset of T1DM. Most of these loci harbour multiple genes and are labelled insulin-dependent diabetes mellitus 1 (IDDM1) to 18 (IDDM18). The two most researched loci, IDDM1 and IDDM2, contain genes that encode immune response proteins and preproinsulin, respectively.63 Currently, the sole treatment option for diabetic patients is insulin administration. However, exogenous insulin is not physiologically regulated. Moreover, the occasional lack of patient adherence exacerbates the condition, resulting in suboptimal control and secondary complications.64 These setbacks can be avoided with gene therapy; nevertheless, ectopic insulin expression alone cannot cure T1DM unless it is accompanied by a regulatory system that responds to variations in glucose concentration in the blood.65 In a recent effort to counteract T1DM using gene therapy, Callejas and coworkers66 aimed at increasing the muscular uptake of glucose through the coexpression of insulin and glucokinase (GK) in skeletal muscle cells. Insulin was required to ensure transport of glucose by insulin-stimulated glucose transporter 4 (GLUT4) and GK was made available to facilitate glucose uptake by muscle cells in the event of hyperglycaemia (high blood glucose). Five beagle dogs induced with streptozotocin and alloxan to exhibit symptoms of T1DM were injected in the leg with adeno-associated viral vectors that carried either an insulin- or a GK-encoding gene. The immunohistochemical analysis and radioimmunoassay of insulin in conjunction with the measurement of blood glucose using a Glucometer Elite analyser (Bayer, Leverkuser, Germany) indicated the normalization of insulin and glucose levels. Further, northern blot analysis confirmed the expression of the two transgenes in the skeletal muscle biopsies of the models. The long-term efficacy of this approach was established as the models, so far, have lived in good health for more than four years.

The prospects of gene therapy go well beyond the current scope of its applications. Researchers are constantly pursuing new strategies that tap into the therapeutic potentials of various cellular components and machineries. Pseudogenes, for instance, can be thought of as suitable tools for gene therapy. In simple terms, a pseudogene is a (usually) non-functional DNA sequence that bears a resemblance to one or two functional parent genes in the genome. The lack of function is because of either the presence of mutations in the pseudogene or the loss of regulatory sequences, resulting in the disruption of transcription or translation.67,68 Dewannieux et al.69 showed that the human endogenous retrovirus HERV-K (HML2) (a non-functional remnant of ancestral retroviral infections) could regain its infectiousness and repeat its life cycle following a multistep recombination event with functional alleles.69 This suggests the possibility of resuscitating beneficial pseudogenes such as ψ connexin 43 (ψCx43), which tends to suppress the growth of breast cancer cells,70 for therapeutic purposes.

Arguments: for and against

Although everyone more or less acknowledges the tremendous potential of gene therapy to revolutionize clinical sciences, some sceptics with backgrounds in both science and ethics have raised questions that not only pose a challenge to its technical aspects but also underline the moral issues provoked by gene therapy. The vast majority of these questions concern the germline mode of gene therapy, as its outcomes are, contrary to the somatic mode, not confined to the patient under treatment. Yet some of the opponents even revile somatic gene therapy, despite its wide public acceptance.

Among the critics, some have gone so far as to argue against gene therapy in its entirety. One of their arguments is that gene therapy is still in its infancy, with a scope that is too limited to overcome its limitations and prevent its possible adverse consequences. These consequences usually arise from flawed gene transfer that may result in mutations (insertional mutagenesis), up-regulation of oncogenes or inactivation of tumour suppressor genes.71 In addition, the present shortcomings of gene therapy are pointed out as major obstacles to its success. Some of the most common genetic disorders such as T1DM, hypertension and Alzheimer’s disease are polygenic in nature. Such disorders, unlike their monogenic counterparts, are extremely complicated to treat effectively and permanently using gene therapy.72 The opponents also assert that the very high cost of gene therapy renders it against fairness and equality, as it would allow only the affluent minority to benefit from treatment.73 Yet another concern raised is that altering the human genome, which is perceived as the blueprint of life, in any form or fashion is an act of ‘playing God’, with serious threats to human dignity.74

As for somatic gene therapy in particular, its opponents express their fear of the probable technical errors and the slippery slope that could lead to crossing the barrier between the somatic mode and the germline mode of therapy. From a technical point of view, they state that the incorrect choice of vectors and target cells could pave the way for a therapeutic transgene to spread to the gonads and affect the genotype of the offspring.73 Socioethically, they claim that permitting somatic gene therapy is a preliminary step on a slippery slope towards what is described as the horrendous manipulation of humankind that is made possible by germline gene therapy.75

When it comes to germline gene therapy, the debate get more intense and the naysayers’ tone more resentful. Their argument is essentially based upon the consequences of deviating from a treatment-centred use of the therapy. The most frequently mentioned concern is employing the method for genetic enhancement. This is often illustrated with healthy individuals such as athletes who seek physiological improvements6 and ‘designer babies’ possessing their parents’ favourite traits.76 Such activities may, as the commentators suggest, set in motion a chain of events leading to the re-emergence of eugenics. Another concern surrounding germline gene therapy is that any accidental mistakes in the process will undoubtedly create a lineage of people whose genomes have been not only manipulated without their consent but also disrupted before they were born.77

On the other hand, the pro-gene therapy arguments hail the technique as a highly efficient and absolutely ethical saviour of human beings in the face of otherwise incurable diseases. People holding this perspective view gene therapy as a continuation of other conventional therapies with bigger, life-saving promises and exaggerated drawbacks. They contend that germline gene therapy is exceptionally efficient, as its application in one individual can preclude the reappearance of a disorder in succeeding generations. Evidently, it would also be less costly for patients and useful in conserving future health care resources.73,78 Furthermore, the advocates put forth the rationale that gene therapy is necessary owing to its ability to cure unique diseases such as Parkinson’s disease79 and retinoblastoma80 that other therapies have consistently failed to beat. On ethical grounds, gene therapy is considered pro-life by the likes of French Anderson, a leading National Institutes of Health investigator, who believes ‘it would be unethical to delay human trials’, as he thinks that ‘the patients have little other hope’.81 In addition, germline gene therapy provides the opportunity for couples to save their genetically challenged embryos, rather than discard them outright.78


The premise of gene therapy as a novel therapeutic technique, capable of introducing new genes and/or correcting existing defective genes in an organism, is as ethically controversial as it is scientifically ground breaking. Both the somatic and germline modes of gene therapy have spurred vehement debates among those who consider it immoral and unreliable and others who admire it on account of its unique and medically plausible potentials. An overall look at the issue and the positions on either side reveals that some aspects or others are being undermined by either the supporters or the critics. On the one hand, those that support gene therapy usually downgrade the fact that it could lead to unexpected repercussions, and, on the other hand, those against gene therapy overstate a few points, such as the lack of morality in modifying genes and the possibility of the non-medical use of gene therapy, without appreciating its very real prospects. First, the notion that altering DNA poses a threat to human dignity is subjective, and may vary from one belief system or theory of ethics to another. This raises the question of whose beliefs should be preferred to those of others who might possibly be in favour of gene therapy. Second, the fear that allowing one form of gene therapy could facilitate the emergence of the other, which in turn could be exploited for non-medical purposes, is merely justified by a rhetorical slippery slope that an action will ultimately culminate in havoc via a chain of uncontrolled events. Although regulation is essential, the slippery slope argument, as it is put forth, would call a halt to most of the ongoing medical and non-medical research across the globe. Third, the fact that gene therapy could be error prone holds true for most scientific developments. However, rigorous research on the technique to verify its safety could significantly minimize the likely errors. In addition, devising and enforcing such regulations as prohibition of non-medical uses of gene therapy, requirement of the patient’s informed consent and proof of the necessity of gene therapy to treat a disease can further help address the issue. In the end, bringing mankind’s creativity, which fuels developments in gene therapy, to a standstill is taking the easy way out, rather than dealing with the issue.



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