Pediatric Nephrology

, Volume 20, Issue 2, pp 118–124

A realistic chance for gene therapy in the near future


    • Department of Pediatrics and Genetic MedicineWeill Medical College of Cornell University
Editorial Commentary

DOI: 10.1007/s00467-004-1680-0

Cite this article as:
Worgall, S. Pediatr Nephrol (2005) 20: 118. doi:10.1007/s00467-004-1680-0


The expanding knowledge of the genetic and cellular mechanisms of human diseases in the post-genomic era coupled with the development of different vector systems to efficiently transfer genes to a variety of cell types and organs in vivo gave rise to the concept of gene therapy as a promising therapeutic option for genetic and acquired diseases. Gene therapy has been the focus of both enthusiasm and critique in the past years. Major progress has been achieved in evaluating gene therapy in clinical trials. However, a number of hurdles must still be overcome to make gene therapy safe and applicable for human diseases. Increased knowledge of the interaction of the gene therapy vehicles with the host has resulted in modifications of existing and the development of new vector systems, as well as adjustments of future clinical applications. Adeno-associated virus vectors, retrovirus- and lentivirus-based vectors show great promise for the correction of monogenic diseases. Correction of the genetic defect can be attempted by either in vivo administration to directly target a diseased organ or by administration of ex vivo genetically modified cells, e.g., bone marrow stem cells. The lack of persistent expression and the immune responses of the host have limited the use of adenovirus vectors for the permanent correction of monogenic diseases. However, the ease of production and the number of cell types and organs that can be efficiently infected make adenovirus-based vectors a promising tool for applications where permanent gene expression is not the therapeutic goal or where the induction of immune responses is the desired response, as for genetic vaccines. Overall, gene therapy remains promising for the correction of genetic as well as acquired disorders, where permanent or transient expression of a gene product will be therapeutic.


Gene therapyGene transfer vectorsClinical trials


Gene therapy is a clinical strategy using the transfer of genes for therapeutic purposes. It is based on the concept that the expression of a transferred gene will result in a modified phenotype in the targeted cells or organ [1, 2]. Gene therapy was initially designed to permanently correct a single gene in monogenetic disorders, such as cystic fibrosis, severe combined immunodeficiency (SCID), or muscular dystrophy [3]. However, targets for gene therapy have expanded to include approaches to eliminate or modify malignant cells, to modify host defenses, or to re-engineer diseased organs, utilizing genes with a role in organ development. Significant research efforts have been invested since the first patients were treated with gene therapy more than a decade ago, and major advances have been accomplished in the development and optimization of gene transfer vector systems [4]. The consequences of the interaction of gene therapy vehicles and the transferred genes with the host have been closely monitored in clinical trials and are an important area of investigation for the design of future gene therapy studies [5]. The two most serious adverse events in humans have been observed in clinical gene therapy studies with adenovirus and retrovirus-mediated gene transfer vectors [6, 7]. A major negative consequence of the use of adenovirus-based vectors is the interaction with the host immune system [5, 8]; integration of retroviral vectors into host DNA has led to the development of malignancies [9, 10]. In spite of these complications, gene therapy remains a viable and promising therapeutic concept, especially in the post-genomic era, following the decoding of the human genome and the rapid discovery of gene alterations as the origin of human diseases.

The kidney, with its variety of inherited disorders, has been a difficult target for gene therapy vectors and has received less attention overall [11]. Although clinical trials to correct inherited renal or urogenital disorders using gene therapy have not yet been performed, research in this area is active and will profit from the progress in the development of new and improved vector systems for other target organs [11, 12, 13, 14]. This review focuses on the major gene transfer vector systems, especially in relation to their application in human disease.

Gene therapy vectors

To facilitate the transfer of genes into cells the genes need to be packaged into vectors. A vector is a carrier that helps to circumvent the natural barriers to DNA internalization to the cell nucleus, where it can use the cellular machinery to express the exogenous gene. In general, gene transfer vector systems can be categorized into viral and non-viral systems (Table 1). Given the wide diversity of potential target organs and cells for gene therapy, a single vector system is unlikely to be suitable for all potential applications.
Table 1

Characteristics of most commonly used gene transfer vectors

Vector type

Transgene capacity




<10 kba

Easy to manipulate

Poor transduction efficiency


Limited persistence

Target cell not specific


7.5 kbb

Easy to produce at high titers

Host immune and inflammatory response

High expression levels

Transient expression

Almost all cell types transducible

Genome not stable

Infects replicating and non-replicating cells

Cell targeting possible

Adeno-associated virus

4.5 kb

Almost no immunogenicity

Difficult to produce at high titers

Non-pathogenic in humans

Small transgenes

Infects replicating and non-replicating cells

Late onset of expression

Site-specific, stable integration

Long-term expression


8 kb


Insertional mutagenesis

Stable integration in dividing cells

Low titers

Cell type retargeting possible

Infects only proliferating cells


8 kb


Safety concerns regarding HIV

Stable integration

Possible mutagenesis

Herpes simplex

10–40 kbc

Good persistence in selected cell-types

Toxicity and immunogenicity

Large expression casette

aPlasmid size is generally not limited, plasmids <10 kb are generally not suitable for gene transfer

bCapacity for helper-dependent (gutless) vectors containing no viral genes is approximately 36 kb

cHerpes virus vectors 10 kb, herpes virus replicons 40 kb

Non-viral vectors

Non-viral vector systems include DNA plasmids or plasmids combined with chemically synthesized vehicles such as cationic liposomes. There has been some success in transferring genes to liver and lung using liposomes; however, the efficiency of gene transfer for these vector systems is very low [15]. The advantage of non-viral vector systems is that they do not activate the host inflammatory and immune response, as do some of the viral vector systems. The mechanism of gene transfer is poorly understood and depends on lipid composition, complex charge density, and the size of the complex [16]. Once the liposome enters the cell, its efficiency in entering the nucleus is significantly less compared with virus-based systems. Another disadvantage is the limited persistence of the transgene expression.

Viral vector systems

The main groups of viral gene transfer vector systems are derived from adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, and herpes simplex virus (HSV). The characteristics of each of these viral vectors make them more or less appropriate for specific applications. These include immunogenicity, vector tropism, persistence of the transgene expression, maximum size of gene that can be packaged, and integration into the host genome leading to persistence in dividing cells [2, 4, 5, 17, 18]. Numerous other viral vector systems have been developed and tested in vitro or in experimental animals, but will not be discussed in this review as they have not yet been used clinically.


Adenoviruses are non-enveloped icosahedral DNA viruses with a 36-kb genome [19]. Deletion of essential genes, typically E1, renders the adenovirus replication deficient and makes room for the insertion of an expression cassette. Adenovirus-based vectors have been most extensively evaluated in clinical studies [20]. The advantages of utilizing adenovirus vectors for gene therapy are that they are easy to propagate in high titers, they can infect most cell types, and they can be manipulated to accommodate large DNA inserts. The first clinical study using adenovirus to correct a genetic disease administered an adenovirus vector encoding the gene for cystic fibrosis transmembrane regulator (CFTR) to the lungs of patients with cystic fibrosis [21]. The resultant level of expression of the CFTR gene at the site of vector deposition was approximately 5%, a level believed to be sufficient for a therapeutic effect. The administration of the vector resulted only in expression for a few days. Although not observed in this study administration of adenovirus vector leads to significant immune responses against adenovirus, reflected in either neutralizing antibodies or adenovirus-specific T cell proliferation [5, 8, 22]. Ornithine transcarbamylase (OTC) deficiency, a recessive metabolic disorder of nitrogen metabolism, is the only other metabolic inherited disease in which adenovirus vectors have been used for gene therapy [23]. Following intravenous administration to target the liver, it became apparent that a large dose of adenovirus vector can be fatal [7]. Another area of investigation is the use of adenovirus vectors to target cancer. These studies are based on the following strategies: local prodrug activation, tumor suppressor genes, immunotherapy, and oncolytic viruses [24, 25, 26]. For the prodrug strategy, the idea is to deliver the gene for an enzyme that activates a prodrug into an active chemotherapeutic agent. Local activation of the prodrug in the tumor will concentrate the active agent in the tumor and thus limit the systemic toxicity from the active drug. A variety of tumor suppressor genes have been used in human clinical studies (p53, mda7, and p16), with the concept that tumor cells have defective tumor suppressor genes that cannot limit cell division, but restoration of the wild type gene will normalize cell division. Immunostimulatory genes, including CD40 ligand and interleukin-2 among others, have been evaluated for the treatment of cancer in clinical studies.

Overall, first-generation adenovirus vectors have a great potential for therapies where only a limited duration of gene expression is necessary and permanent expression of the transferred gene is not needed and may lead to undesired effects. An application that fits these criteria is the induction of angiogenesis. Therapy of coronary artery and peripheral vascular disease has used the genes for angiogenic growth factors such as VEGF [27, 28]. Temporary expression of VEGF leads to angiogenesis and finally to vascularization of the underperfused area, whereas prolonged expression may induce uncontrolled vessel growth with the potential for angiogenesis in distant areas. Another application for adenovirus vectors, where long-term expression is not desirable and the induction of immune responses is wanted, is as a vaccine vehicle [29]. Based on their strong immunogenic and inflammatory properties, adenovirus vectors act as a powerful adjuvant for a variety of antigens and are currently being tested as vaccine carriers against HIV and Ebola [30, 31]. For this application, manipulation of the adenovirus vectors, e.g., to target antigen-presenting cells more efficiently, may increase their efficiency as a vaccine [32]. Various attempts have been made to change the immunogenicity of the first-generation adenovirus vectors by deleting more genes from their adenovirus genome by creating helper-dependant (“gutless”) vectors [33]. The helper-dependant adenovirus vectors contain no adenovirus genome and demonstrate less immunogenicity and longer persistence in animals [34]. These vectors have so far not been tested in larger clinical trials.

Adeno-associated virus

Adeno-associated virus (AAV) is a member of the parvovirus family. The wild type AAV genome is 4.7 kb and integrates into the genome at a specific site on chromosome 19 [35]. No human disease has been associated with AAV infection. There are at least seven serotypes known. Recently there have been additional types recovered from mammalian tissue samples and some are currently being analyzed for their potential use as vectors [36, 37]. Different serotypes infect tissues with varying efficiency. The serotype most commonly used for AAV vectors is serotype 2, which infects brain and retina very well, and thus has been used for a wide spectrum of applications targeting these organs [38, 39]. AAV1 infects muscle well. As more and more AAV serotypes are being characterized and different serotype capsids can also be combined to generate novel tropisms, there is the potential that tissues that have not been easily infected with the current AAV serotypes may become amenable to AAV-based gene transfer in the future. A number of clinical trials evaluating the use of AAV vectors for genetic and acquired diseases are currently under way. The brain is the target for monogenetic metabolic disorders, such as Canavan disease [40] or degenerative disorders such as Parkinson disease [41]. AAV-mediated transfer of factor IX to the muscle or liver is currently being investigated as a therapy for hemophilia B. Administration of genes encoding secreted proteins to the muscle for systemic delivery is an attractive application for AAV-mediated gene transfer [42].

Retrovirus and lentivirus

Vector systems based on retrovirus and lentivirus have sufficient space for many applications (8 kb), have low immunogenicity, and lead to persistent gene expression [17]. Retrovirus vectors were the first viral vector system to be used for gene therapy. As retroviruses integrate into the genome of dividing cells, the most promising target is rapidly dividing hematopoietic cells. The main targets for retroviral vectors are inherited disorders of T-cells and hematological malignancies. To date, the only significant correction of a genetic disorder using gene therapy is the retrovirus-mediated gene transfer to hematopoietic stem cells [43, 44].

Lentiviral vectors can infect dividing and non-dividing cells, which broadens their potential applications to target cell types not able to be infected with retroviral vectors [45]. Studies in animals have shown successful correction of genetic hematological disorders such as thalassemia using gene transfer to hematopoietic stem cells [46] and have shown successful gene transfer to brain and liver [45]. Lentiviral vectors are the newest member of the gene transfer vectors and to date there has been one clinical trial using a lentiviral vector in hematopoietic cells of HIV patients initiated in the United States. As with retrovirus vectors, insertional mutagenesis is a potential risk. Recently, a tendency for retroviral or lentiviral vectors to integrate in or near transcriptionally active genes has been reported and various approaches, such as the use of genetic insulators and cell-specific regulatory elements, are being developed to decrease this risk [47, 48, 49].


Vectors derived from HSV have been evaluated, especially for gene therapy of neuronal cells [18]. HSV vectors have the largest capacity of all viral vector systems described to incorporate a transgene. However, the toxicity and inflammation observed in vivo have limited their use [50].

Problems and hurdles

Several challenges have to be overcome for gene therapy to be successful. These include the safety of recombinant DNA technology in humans, cell and tissue targeting, the host immune response against the transferred gene product and the vector system, as well as the regulation of gene expression [5]. Each of these has been the subject of intense investigation. The safety of recombinant DNA technology will ultimately have to be determined in well-controlled and well-monitored human trials. To date more than 600 clinical gene therapy trials have been initiated [5, 50]. The first successful trial to correct a genetic disease using gene therapy was with retrovirally transduced CD34+ hematopoietic stem cells in children with SCID. Immune parameters of these children normalized [44], however insertional mutagenesis resulting in expression of the oncogene gene LMO2 occurred and lead to T-cell acute leukemia-like syndrome in two of the patients [9]. One patient in a study using an adenovirus vector expressing OTC to correct OTC deficiency suffered a fatal fulminant inflammatory response [7]. Based on this experience, gene therapy trials involving adenovirus vectors were temporarily halted [51]. However, the safety profile of adenovirus vectors in other trials has been good [52]. The induced immune and inflammatory responses are a concern, especially for adenovirus- and HSV-based vectors [8, 50]. Antigens present in the viral vector and the expression of the transgene both cause cellular and humoral immune responses, which depend on the viral vector, the route of administration, and the genotype and infection history of the host [8]. Immunity against the transferred gene product may be a concern for monogenic disorders where the host has never seen the protein before and has thus not been tolerized. There seem to be differences based on the genetic disease and the site of vector administration with regard to the immunity against the transgene; these are not fully understood and have been observed in animal studies [53]. The experience in human studies is limited. No immune response against α1-antitrypsin was observed in patients with α1-antitrypsin deficiency receiving gene therapy vectors expressing α1-antitrypsin [54]. In contrast, gene transfer with factor IX to correct hemophilia [55] seems to induce more immune responses against the transgene [56, 57]. These findings mirror the experiences with protein replacement therapy for these factors. One potential strategy to circumvent the immune response against the vector and/or transgene may be to use immunosuppression, which has been shown to be effective when administered for a short period following vector administration [58]. The negative consequences of immunosuppression have obviously to be carefully weighed against the benefits of the gene correction.

Gene therapy for kidney disease

Gene transfer to the kidney has been problematic for a variety of reasons, but has been accomplished with liposomal, AAV, adenovirus, and lentiviral vectors using the vascular, urethral, and direct injection route in animal studies [11, 12, 13, 14, 59]. Potential applications for gene therapy include acquired, immune-mediated disorders, as wells as inherited diseases such as Alport syndrome or polycystic kidney disease. In addition, up to two-thirds of children with chronic renal insufficiency suffer from congenital abnormalities of the kidney and the urinary tract. It is likely that most of these abnormalities are genetically determined, and mutations in a variety of developmentally active genes have been described. The identification of genetic factors involved in the development of these abnormalities will aid in the design of studies using genetic modification, although most of these disorders may not be monogenetic and have a variable phenotype. Bone morphogenetic protein-4, for example, could be a potential candidate for genetic manipulation, as it has been shown to prevent cell death and promote the growth of metanephric mesenchyme in a mouse model [60].

As the kidney is composed of a variety of cell types, targeting a specific cell type may be necessary and has been problematic if corrective gene therapy is being attempted via the vascular route. An alternative approach is the intravascular administration of ex vivo genetically modified cells, e.g., genetically modified macrophages or bone marrow progenitors to target glomerular or interstitial disease [59, 61, 62]. With the advances in stem cell technology, genetic modification of stem cells or mobilization and recruitment of stem cells will be an attractive strategy for the gene therapy of renal diseases [63, 64, 65]. Transferring genes encoding soluble proteins, such as a soluble inhibitor of transforming growth factor-β to influence renal fibrotic disease, to a distant, more readily accessible site such as muscle has been shown to positively affect renal disease in animal studies [66]. A promising target for gene therapy of kidney might be the transplant kidney, which can be genetically modified ex vivo. Various strategies for gene transfer to the transplant kidney have been investigated to target rejection, allograft nephropathy, and ischemia reperfusion [67, 68, 69].


Gene therapy has come a long way, but after more than a decade of intense research significant challenges still exist before gene therapy can become an established therapy for human disease. The potential applications for gene therapy have broadened since the original concept of its use for the correction of monogenetic disorders. Strategies to combine stem cell approaches with genetic modification are an exciting new area of investigation. The improvement in the existing and the development of new vector systems will aid the tailored application of gene therapy strategies for a variety of acquired and inherited diseases. The efficacy and safety of gene therapy strategies, especially the interaction of the vector systems with the host, need to be vigorously assessed in preclinical studies, but ultimately gene therapy strategies to treat human diseases can only be evaluated by performing carefully designed clinical trials.


I thank Anja Krause for helpful discussions and critical review of the manuscript.

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© IPNA 2004