Gene therapy is the introduction of a biologically active gene into a cell to achieve a therapeutic benefit. In 2012, the first gene therapy product was licensed in the United States and Europe for the treatment of lipoprotein lipase deficiency, and gene therapy has now been approved for the treatment of several more disorders; the number in late-stage clinical trials exceed a dozen, some of which are outlined in Table 1. These recent successes firmly establish that the treatment of genetic disease at its most fundamental level – the gene – will be increasingly feasible. The goal of gene therapy is to transfer the therapeutic gene early enough in the life of the patient to prevent the pathogenetic events that damage cells. Moreover, correction of the reversible features of genetic diseases should also be possible for many conditions.

Table1. Examples of Inherited Diseases Treated by Gene Therapy of Somatic Tissues

In this section, we outline the potential, methods, and probable limitations of gene transfer for the treatment of human genetic disease. The minimal requirements that must be met before the use of gene transfer can be considered for the treatment of a genetic disorder are presented.

General Considerations for Gene Therapy

In the treatment of inherited disease, the most common use of gene therapy will be the introduction of functional copies of the relevant gene into the appropriate target cells of a patient with a loss-of-function variant (because most genetic diseases result from such variants).

In these instances, precisely where the transferred gene inserts into the genome of a cell would, in principle, generally not be important. If gene editing to treat inherited disease becomes routinely possible, then correction of the defect in the mutant gene in its normal genomic context would be ideal and would alleviate concerns such as the activation of a nearby oncogene by the regulatory activity of a viral vector or the inactivation of a tumor suppressor due to insertional mutagenesis by the vector. In some long-lived types of cells, stable, long-term expression may not require integration of the introduced gene into the host genome. For example, if the transferred gene is stabilized in the form of an episome (a stable nuclear but nonchromosomal DNA molecule, such as that formed by an adeno associated viral vector, discussed later), and if the target cell is long-lived (e.g., T cells, neurons, myocytes, hepatocytes), then long-term expression can occur without integration.

Gene therapy may also be undertaken to inactivate the product of a dominant mutant allele whose abnormal product causes the disease. For example, vectors carrying siRNAs (see earlier section) could, in principle, be used to mediate the selective degradation of a mutant mRNA encoding a dominant negative proα1(I) collagen that causes osteogenesis imperfecta.

Gene Transfer Strategies

An appropriately engineered gene may be transferred into target cells by one of two general strategies (Fig. 1). The first involves introduction of the gene into cells that have been cultured from the patient ex vivo (i.e., outside the body) and then reintroduction of the cells to the patient after the gene transfer. In the second approach, the gene is injected directly in vivo into the tissue or extracellular fluid of interest (from which it is taken up by the target cells). In some cases, it may be desirable to target the vector to a specific cell type; this is usually achieved by modifying the coat of a viral vector so that only the designated cells bind the viral particles.

Fig1. The two major strategies used to transfer a gene to a patient. For patients with a genetic disease, the most common approach is to construct a viral vector containing the human complementary DNA (cDNA) of interest and to introduce it directly into the patient or into cells cultured from the patient that are then returned to the patient. The viral components at the ends of the molecule are required for the integration of the vector into the host genome. In some instances, the gene of interest is placed in a plasmid, which is then used for the gene transfer.

The Target Cell

 The ideal target cells are stem cells (which are self replicating) or progenitor cells taken from the patient (thereby eliminating the risk for graft-versus-host disease); both cell types have substantial replication potential. Introduction of the gene into stem cells can result in the expression of the transferred gene in a large population of daughter cells. At present, bone marrow is the only tissue whose stem cells have been successfully targeted as recipients of transferred genes. Genetically modified bone marrow stem cells have been used to cure two forms of SCID, as dis cussed later. Gene transfer therapy into blood stem cells is also likely to be effective for the treatment of hemoglobinopathies and storage diseases for which bone marrow transplantation has been effective, as discussed earlier.

An important logistical consideration is the num ber of cells into which the gene must be introduced to have a significant therapeutic effect. To treat PKU, for example, the approximate number of liver cells into which the phenylalanine hydroxylase gene would have to be transferred is ~5% of the hepatocyte mass, or ~1010 cells, although this number could be much less if the level of expression of the transferred gene is higher than wild-type. A much greater challenge is gene therapy for muscular dystrophies, for which the gene must be inserted into a significant fraction of the huge number of myocytes in the body to have therapeutic efficacy.

DNA Transfer Into Cells: Viral Vectors

The ideal vector for gene therapy would be safe, readily made, and easily introduced into the appropriate target tissue, and it would express the gene of interest for life. Indeed, no single vector is satisfactory in all respects for all types of gene therapy, and a repertoire of vectors is required. Here, we briefly review three of the most widely used classes of viral vectors, those derived from retroviruses, adeno-associated viruses (AAVs), and adenoviruses.

One of the most widely used classes of vectors is derived from retroviruses, simple RNA viruses that can integrate into the host genome. They contain only three structural genes, which can be removed and replaced with the gene to be transferred (see Fig. 1). The current generation of retroviral vectors has been engineered to render them incapable of replication. In addition, they are nontoxic to the cell, and only a low number of copies of the viral DNA (with the transferred gene) integrate into the host genome. Moreover, the integrated DNA is stable and can accommodate up to 8 kb of added DNA, commodious enough for many genes that might be transferred. A major limitation of many retroviral vectors, however, is that the target cell must undergo division for integration of the virus into the host DNA, limiting the use of such vectors in nondividing cells such as neurons. In contrast, lentiviruses, the class of retroviruses that includes HIV, are capable of DNA integration in nondividing cells, including neurons. Lentiviruses have the additional advantage of not showing preferential integration into any specific gene locus, thus reducing the chances of activating an oncogene in a large number of cells.

AAVs do not elicit strong immunologic responses, a great advantage that enhances the longevity of their expression. Moreover, they infect dividing or nondividing cells to remain in a predominantly episomal form that is stable and confers long-term expression of the transduced gene. A disadvantage is that the current AAV vectors can accommodate inserts of up to only 5 kb, which is smaller than many genes in their natural context.

The third group of viral vectors, adenovirus-derived vectors, can be obtained at high titer, will infect a wide variety of dividing or nondividing cell types, and can accommodate inserts of 30 to 35 kb. However, in addition to other limitations, they have been associated with at least one death in a gene therapy trial through the elicitation of a strong immune response. At present their use is restricted to gene therapy for cancer.

Risks of Gene Therapy

Gene therapy for the treatment of human disease has risks of three general types:

• Adverse response to the vector or vector-disease com bination. Principal among the concerns is that the patient will have an adverse reaction to the vector or the transferred gene. Such problems should be largely anticipated with appropriate animal and preliminary human studies.

• Insertional mutagenesis causing malignancy. The second concern is insertional mutagenesis – that is, the transferred gene will integrate into the patient’s DNA and activate a protooncogene or disrupt a tumor suppressor gene, leading possibly to cancer. The illicit expression of an oncogene is less likely to occur with the current generation of viral vectors, which have been altered to minimize the ability of their promoters to activate the expression of adjacent host genes. Insertional inactivation of a tumor suppressor gene is likely to be infrequent and, as such, is an acceptable risk in diseases for which there is no therapeutic alternative.

• Insertional inactivation of an essential gene. A third risk – that insertional inactivation could disrupt a gene essential for viability – will, in general, be without significant effect because such lethal mutations are expected to be rare and will kill only single cells. Although vectors appear to somewhat favor insertion into transcribed genes, the chance that the same gene will be disrupted in more than a few cells is extremely low. The one exception to this statement applies to the germline; an insertion into a gene in the germline could create a dominant disease-causing mutation that might manifest in the treated patient’s offspring. Such events, however, are likely to be rare and the risk acceptable because it would be difficult to justify withholding, on this basis, carefully planned and reviewed trials of gene therapy from patients who have no other recourse. Moreover, the problem of germline modification by disease treatment is not confined to gene therapy. For example, most chemotherapy used in the treatment of malignant disease is mutagenic, but this risk is accepted because of the therapeutic benefits.

Diseases That Have Been Amenable to Gene Therapy

 More than two dozen single-gene diseases have been shown to improve with gene therapy, and a large number of other monogenic disorders are potential candidates for this strategy, including retinal degenerations, hematopoietic conditions such as sickle cell anemia and thalassemia, and disorders affecting liver proteins such as PKU, urea cycle disorders, familial hypercholesterolemia, and α1AT deficiency. Here we discuss several dis orders in which gene therapy has been clearly effective, and we highlight some of the challenges associated with this therapeutic approach.

X-linked Severe Combined Immunodeficiency

The SCIDs are due to pathogenic variants in genes required for lymphocyte maturation. Affected individuals fail to thrive and die early in life of infection because they lack functional B and T lymphocytes. The most common form of the disease, X-linked SCID, results from variations in the X-linked gene IL2RG, encoding the γc-cytokine receptor subunit of several interleukin receptors. The receptor deficiency causes an early block in T- and natural killer–lymphocyte growth, survival, and differentiation and is associated with severe infections, failure to thrive, and death in infancy or early childhood if left untreated. This condition was chosen for a gene therapy trial for two principal reasons. First, bone marrow transplantation cures the disease, indicating that the restoration of lymphocyte expression of IL2RG can reverse the pathophysiologic changes. Second, it was believed that transduced cells carrying the transferred gene would have a selective survival advantage over untransduced cells.

The outcome of trials of X-linked SCID has been dramatic and resulted, in 2000, in the first gene therapy cure of a patient with a genetic disease. Subsequent con firmation has been obtained in most patients in subsequent clinical trials (see Table 1). Bone marrow stem cells from the patients were infected in culture (ex vivo) with a retroviral vector that expressed the γc cytokine subunit cDNA. A selective advantage was conferred on the transduced cells by the gene transfer. Transduced T cells and natural killer cells populated the blood of treated patients, and the T cells appeared to behave normally. Although the frequency of transduced B cells was low, adequate levels of serum immunoglobulin and antibody levels were obtained. Dramatic clinical improvement occurred, with resolution of protracted diarrhea and skin lesions and restoration of normal growth and development. These initial trials demonstrated the great potential of gene therapy for the correction of inherited disease.

This highly promising outcome, however, came at the cost of induction of a leukemia-like disorder in 5 of the 20 treated patients, who developed an extreme lymphocytosis resembling T-cell acute lymphocytic leukemia; 4 of them are now well after treatment of the leukemia. The malignancy was due to insertional mutagenesis: The retroviral vector inserted into the LMO2 locus, causing aberrant expression of the LMO2 mRNA, which encodes a component of a transcription factor complex that mediates hematopoietic development. Consequently, trials using integrating vectors in hematopoietic cells must now monitor insertion sites and survey for clonal proliferation. Current-generation vectors are designed to avoid this mutagenic effect by using strategies such as including a self-inactivating or “suicide” gene cassette in the vector to eliminate clones of malignant cells. At this point, bone marrow stem cell transplantation remains the treatment of choice for those children with SCID fortunate enough to have a donor with an HLA-identical match. For patients without such a match, autologous transplantation of HSPCs, in which the genetic defect has been corrected by gene therapy, offers a lifesaving alternative, but one that may not be without risk.

Spinal Muscular Atrophy

 SMA is a monogenic defect associated with the loss of SMN protein and became an excellent candidate for gene replacement therapies when it was discovered that systemic delivery of AAV9-based gene transfer via intravenous injection can cross the blood-brain barrier and efficiently transform target cells in the central nervous system, including motor neurons in the spinal cord in mice and nonhuman primates. This changed the assumption that neurologic disorders may not be candidates for gene therapies. Development of the self-complementary AAV9 (scAAV9) vector further improved the efficiency and speed of gene transcription in a number of preclinical experiments. The first gene therapy trial for SMA included 15 infants, 3 with low dose and 12 with high dose. All 15 patients survived to 20 months and did not require respiratory support. Eleven patients reached the motor milestone of sitting unassisted, and two patients were able to walk independently. These data plus additional data released from the successive phase II/III trial led to FDA approval in 2019. The main benefits of this method are that a single, one-time injection is required, and the SMN protein becomes systemically expressed. Safety and tolerability, however, have to be strictly monitored as acute hepatotoxicity and sensory neuron toxicity were reported in primates and piglets following high-dose intravenous administration of AAV vectors expressing human SMN. As a result, most individuals require prednisolone therapy to minimize the hepatotoxicity. Another consideration is the reported presence of preexisting anti-AAV9 antibodies in SMA patients, which might influence tolerability and efficacy.

RPE65-associated Retinal Dystrophy

Leber congenital amaurosis (LCA), an autosomal recessive condition, comprises a heterogeneous group of eye diseases that cause nystagmus and significant visual impairment in early infancy and total blindness by the third to fourth decades of life. LCA2 (also known as RPE65-LCA) is associated with pathogenic variants of the RPE65 gene encoding the retinoid isomerohydrolase in the retinal pigment epithelium (RPE), which result in rod-cone–type retinal dystrophy. The gene therapy can be injected directly into the eye’s subretinal space, which is an immunoprivileged organ. The majority of studies are conducted on one eye, while the other serves as a control. For RPE65-associated retinal dystrophy voretigene neparvovec is a gene therapy that packages the RPE65 cDNA in an AAV2 vector. This therapy was approved by the FDA in 2017. Follow-up of the original group through 4 years has shown sustained benefit, and additional gene therapy trials are currently underway to treat several other types of LCA.

Hemophilia B

 Hemophilia B is an X-linked disorder of coagulation caused by pathogenic variants in the F9 gene, leading to a deficiency or dysfunction of clotting factor IX. The disease is characterized by bleeding into soft tissues, muscles, and weight-bearing joints and occurs within hours to days after trauma. Severely affected subjects, with less than 1% of normal levels of factor IX, have frequent bleeding that causes crippling joint disease and early death. Prophylactic – but not curative – treatment with intravenous factor IX concentrate several times a week is expensive and leads to the generation of inhibitory antibodies.

In 2011, the first successful gene therapy treatment of hemophilia B was reported in six patients using an AAV8 vector that is tropic for hepatocytes, where fac tor IX is normally produced. After a single infusion of the AAV8-F9 vector, four patients were able to dis continue prophylactic factor IX infusions, whereas the other two tolerated longer intervals between infusions. The two patients who received the highest dose of the vector had transient asymptomatic increases in liver enzyme levels – which resolved with steroid treatment – indicating that immune-related side effects must remain a concern in future studies. Unfortunately, the AAV vectors cannot accommodate the gene for factor VIII, so other vectors will have to be developed for hemophilia A patients. Apart from this limitation of cargo size, however, AAV-mediated gene therapy targeted to hepatocytes may be applicable to any genetic disease in which production of the protein in the liver is the desired goal.

The Prospects for Gene Therapy

To date, more than 5000 clinical gene therapy trials (the majority are for cancer) have been undertaken worldwide to evaluate both the safety and efficacy of this long-promised and conceptually promising technology. Approximately 775 of these trials were for the treatment of monogenic diseases. The exciting results obtained with gene therapy to date, albeit with small numbers of patients and only a few diseases, validate the optimism behind this immense effort. Although the breadth of applications remains uncertain, it is to be hoped that over the next few decades, gene therapy for both monogenic and genetically complex diseases will contribute to the management of many disorders, both common and rare.

اخر الاخبار

اخبار العتبة العباسية المقدسة

شعبة الخطابة النسوية تستعد لإطلاق مسابقة (سبيل المنتظرين) بنسختها الثالثة

قسم الشؤون الفكرية يقدّم محاضرتين توعويتين لطلبة جامعة المنارة في ميسان

سماحة السيد الصافي يؤكّد أن القرآن عظّم شخصية النبي (صلّى الله عليه وآله) وكشف زيف ما أُلصِق بسيرته

العتبة العباسية المقدسة تفتح باب الاشتراك في مسابقة مراقي المجتبى للقصيدة العمودية السادسة

المزيد

الآخبار الصحية

عرض شائع على الأصابع.. قد يكشف سرطان الرئة مبكرا

7 مشروبات طبيعية لتقليل التوتر والقلق

دراسة: "خطر مبكر" يهدد صحة الأطفال

الجوز أو الفول السوداني.. أيهما يتفوّق في حماية القلب؟

المزيد

الآخبار التكنلوجية

مفاجأة علمية عن الإنجاب.. كم طفلا يطيل عمر المرأة؟

استطلاع يكشف "الأكثر قلقا" من تأثر الوظائف بالذكاء الاصطناعي

هل يمنحك تناول الجزر يوميا سمرة طبيعية؟

إنجاز طبي تاريخي.. جراحة قلب بلا شق في الصدر

المزيد

مواضيع ذات صلة

The Genetics of mtDNA Disease

The APOE Gene Is an Alzheimer Disease Susceptibility Locus

The Genetics of Alzheimer Disease

Molecular Testing for Spinal Muscular Atrophy

Genetics of Spinal Muscular Atrophy

The Phenotypes of Spinal Muscular Atrophy

Chondrodysplasias are Caused by Mutations in Genes Encoding Type II Collagen & Fibroblast Growth Factor Receptors

The Genetics of Osteogenesis Imperfecta

Molecular Abnormalities of Collagen in Osteogenesis Imperfecta

Prenatal Diagnosis of Hereditary Genetic Diseases

Prenatal Diagnosis of Non-hereditary Genetic Diseases

Clinical Applications of Gene Testing in Muscular Dystrophy