Indian Journal of Ophthalmology

: 2002  |  Volume : 50  |  Issue : 3  |  Page : 173--181

Gene therapy in ocular diseases

Vijay K Singh, P Tripathi 
 Department of Immunology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India

Correspondence Address:
Vijay K Singh
Department of Immunology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow


Gene therapy is a novel form of drug delivery that enlists the synthetic machinery of the patient«SQ»s cells to produce a therapeutic agent. Genes may be delivered into cells in vitro or in vivo utilising viral or non-viral vectors. Recent technical advances have led to the demonstration of the molecular basis of various ocular diseases. Ocular disorders with the greatest potential for benefit of gene therapy include hereditary diseases such as retinitis pigmentosa, tumours such as retinoblastoma or melanoma, and acquired proliferative and neovascular retinal disorders. Gene transfer into ocular tissues has been demonstrated with growing functional success and may develop into a new therapeutic tool for clinical ophthalmology in future.

How to cite this article:
Singh VK, Tripathi P. Gene therapy in ocular diseases.Indian J Ophthalmol 2002;50:173-181

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Singh VK, Tripathi P. Gene therapy in ocular diseases. Indian J Ophthalmol [serial online] 2002 [cited 2022 Jul 2 ];50:173-181
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Full Text

The promise of being able to manipulate human genetic material in order to treat diseases has long been a hope for patients with untreatable diseases. Gene therapy, the treatment or prevention of diseases by gene transfer, is recognized as an important scientific achievement of the twentieth century.[1][2][3]

The idea of gene therapy is not as new as it seems. After a decade of long struggle at the laboratory bench and many long hours under the harsh lights of the United States federal review process, gene therapy has emerged as a legitimate scientific discipline. The recombinant DNA process opened possibilities that brought the concepts of "inborn errors of metabolism", "one gene-one enzyme", DNA as "the transforming principle", "molecular disease", and the "double helix", to the bedside of a critically sick little girl. It was on September 14, 1990 that Dr. Culver[4] inserted a needle into the left hand of that child and started the infusion of genetically repaired cells, and defined a fundamental change in the human potential for health. Since that historic day, the use of gene therapy has spread to several types of cancer, cystic fibrosis, and familial hypercholesterolemia.

However, the application of gene therapy in the treatment of ocular diseases presents us with interesting and unique questions. It is still developing, and requires further efforts before it is brought to the clinic.[5] Development of a successful strategy for gene therapy depends on several factors. A clinically appropriate disease must be chosen. The molecular genetic basis of the disease must be understood. A mechanism must be available to deliver the desired gene to the therapeutic site. Finally, the strategy must be designed to ensure expression of the therapeutic gene in the appropriate cells and at the appropriate level.

 Gene Transfer Systems

Gene therapy is based on strategies for delivering genes, which is accomplished by means of gene delivery vehicles known as vectors. These vectors encapsulate therapeutic genes for delivery to cells.[6] Though efficient gene delivery remains a substantial obstacle to widespread human clinical trials, many gene delivery methods are under investigation. These include both viral and non-viral vectors [Table:1].[7] Viral vectors are today's choice for gene insertion as they provide high gene transfer efficiency.[8] Adenovirus, adeno-associated virus (AAV), encapsulated adenovirus mini-chromosomes, herpes simplex virus (HSV) and retroviruses (particularly lentiviruses) are the most frequently used viral vectors to date.[9]

Retroviral vectors have been used extensively in clinical trials related to gene therapy. These vectors have several advantages. Since gene insertion appears to be targeted to the genome, expression is quite stable. Little immune response is generated from cells exposed to these vectors. Retroviral vectors have great potential in disorders such as proliferative vitreoretinopathy, diabetic retinopathy and neovascularization associated with age-related macular degeneration (ARMD) in which unwanted cell proliferation is critical to the pathogenesis of the disorder. Cell division is minimal in the mature human eye, and is limited to discrete cell types. Using retroviral vectors one can selectively transfer genes into the dividing cells and get a therapeutic effect without affecting other normal ocular non-proliferating tissues.[7],[10]

Lentiviral vectors have been studied as potential tools in gene delivery. These vectors offer the potential advantages of limited immune response and delivery of the gene into the host cell genome. Since their first application they have been suitably modified in design, biosafety and the ability of transgene expression into target cells. Gene transfer vectors based on lentiviruses are distinguished by their ability to transduce non-dividing cells. In future, regulated lentiviral vectors may improve the safety and efficacy of gene therapy.[11][12][13][14]

The adenovirus has attracted most attention as a viral vector due to its capability of delivering large amounts of exogenous DNA to cells with subsequently high protein expression.[8] Adenoviral vectors have been used to attempt transferring the gene into retinal cells where gene transfer by a retroviral vector was difficult because of minimal cell division (Figure). These vectors have also been used to transfect a foreign gene into corneal endothelial cells, trabecular meshwork cells and cultured retinal pigment epithelial (RPE) cells.[10]

The ability of an adenoviral vector to transfer in vivo the Escherichia coli lac Z gene into ocular cells of mice and rabbits has been analysed.[15] It was found that injection of up to 3 X 107 PFU (plaque forming units) in mice and 109 PFU in rabbit vitreous cavity, anterior chamber or peribulbar space did not result in any detectable cytopathic effect. It was associated with endocytosis of viral particles in corneal endothelial, photoreceptor, bipolar, ganglionic and occulomotor muscle cells, depending on the route of administration. With the viral titre at 3 X 107 or 109 PFU, the expression was detected for at least 50 days. These results opened the possibility for treatment of retinal hereditary disorders and acquired corneal or retinal inflammatory diseases.

AAV has also received attention for lack of apparent immune response directed against it. It has been reported that recombinant AAV vector can deliver genes into corneal endothelial cells, and transgene expression is dramatically induced by inflammation.[16] The recombinant AAV-delivered transgene was stable and did not compromise endothelial cell function. Thus, inducible recombinant AAV-mediated transgene expression in corneal endothelial cells can be a promising strategy in the treatment and prevention of ocular diseases. However, technical problems continue and its potential use appears uncertain.[8] AAV vectors have been used to transfer the lac Z gene into retinal cells.[10] Similarly, delivery and stable expression of exogenous genes to the retina using lentiviral vectors has also been demonstrated.[17] However, several issues remain to be resolved before lentiviral vectors make it to the bedside.[18] These vectors are limited by relatively poor transduction efficiency as well as the size of DNA they can deliver.

Viral vectors have their own shortcomings. They have the ability to elicit an immune response, and have potential for toxicity. In the recent past, large numbers of non-viral approaches have emerged, yielding safer and promising pre-clinical results. These are currently evaluated in clinical trials.[19] So far, various types of non-viral vectors have been proposed; they can be divided into two broad categories: naked DNA delivery by a physical method, and delivery by a complex of DNA with a cationic carrier. In the former case, DNA can be directly delivered to the cytoplasm. DNA-cationic carrier complex requires an endosomal and/or lysosomal release since it is entrapped in these organelles after its cellular uptake.[20]

 Prospects of Gene Therapy in Ocular Diseases

The eye is one of the most suitable targets for gene therapy. It is easily accessible and allows local application of therapeutic agents with reduced risk of systemic effects.[8],[17] In addition, the effects of treatment may be monitored by a variety of non-invasive examinations. In the eye, the retina is possibly the best candidate for gene therapy. The amount of virus injected into the retina is about 1/1000 of the amount used for systemic diseases. The blood ocular barrier within the eye separates it from the rest of the body, acting to protect the retina and preventing the escape of large molecules into the blood stream. Therefore, a virus delivered to the eye is unlikely to cause any systemic disease.[21] Thus, gene therapy may become a therapeutic modality in the treatment of ocular diseases, in addition to serving as a method for studying mechanisms of the disease pathogenesis.

Gene transfer experiments have demonstrated that it is possible to deliver transgenes to the retina in vivo in stable and efficient fashion with minimal toxicity.[22]There are many types of ocular disorders, which may be amenable to gene therapy. Some of the ocular diseases where gene therapy has been exploited with some success are given below and in [Table:2].

 Retinitis pigmentosa (RP)

RP is a group of hereditary retinal degenerative diseases characterised by a progressive loss of vision due to the degeneration of photoreceptor cells.[23,24] It is considered to be a suitable candidate for gene therapy.[25] The availability of animal models (transgenic and knockout mice), with mutations in the genes for key components of the visual phototransduction (process which turns light into an electrical signal in the brain) cascade, provides a special opportunity and a very powerful research tool. These models are used to investigate the molecular mechanisms of phototransduction cascade and promise to provide valuable information on the pathogenesis of RP.[26],[27] Furthermore, by harnessing therapeutic genes to the viruses, researchers have demonstrated rescue of vision loss in rodent models of RP.[26] It suggests that gene therapy may be effective in delaying photoreceptor cell death.

Researchers are evaluating different approaches to slow RP progression.[9] For the first time, a new treatment called "ribozyme therapy" has been shown to slow down the progress of RP successfully in an animal model. Ribozyme therapy is a new technique that uses specialized molecules (ribozymes), which act as molecular scissors to target the mutant gene products or neurotropins (naturally occurring protein growth factors), which inhibit apoptotic cell death. The ribozyme targets the mutant gene and then "splits" or "cleaves" its mutant messenger RNA (mRNA, the destructive product of mutant genes), to slow or halt the production of destructive protein.[28]

Some investigators have developed transgenic rats that carry gene mutations for dominant forms of RP and have found that ribozyme gene therapy dramatically reduces vision loss in these rats. The photoreceptor cell function was as much as 93% greater in treated eyes than in the untreated controls.[29] Furthermore, when the ribozyme inserted into the AAV vector was injected into the subretinal space, it located and bound to the mutated mRNA of the defective photoreceptor. It cuts the mRNA in half and decreases the quantity of injury causing protein before it can damage the photoreceptor.[30] Investigators have created a ribozyme that recognizes the mRNA for autosomal dominant RP. It encodes the mutant P23H rhodopsin, and destroys it before the rhodopsin makes the photoreceptor-degenerating P23H protein. The photoreceptors survive with the single normal copy of the rhodopsin gene in their other allele.[31]

Another approach has involved injecting rats with vectors carrying neurotrophins. When genes encoding the neurotrophins were added into the virus vector, it was found that these genes prompted the retina to secrete the neurotrophins in perpetuity, and slowed the disease progression by 30 to 40%.[32][33][34]

Liang et al,[35] have investigated intravitreal administration of an AAV carrying ciliary neutrophic factor in animal model of RP. They aimed to study the longterm capabilities of morphological and physiological rescue of photoreceptors and whether administration of this virus is effective in protecting the remaining photoreceptors after the degeneration has set in. They demonstrated that intravitreal administration of recombinant AAV resulted in efficient transduction of retinal ganglion cells.

Investigators have shown that AAV is capable of transducing photoreceptor cells and thus supported the use of this vector system for gene insertion in RP.[36] They further reported that highly purified recombinant AAV vectors including the reporter lac Z gene transduce photoreceptors in an immunocompetent mouse strain following subretinal injection, and efficiently transduce ganglion cells after intravitreal injection.

 Gyrate atrophy

This is a genetic disorder in which patients lack a specific gene, known as ornithine amino transferase (OAT). This rare but well characterized genetic error leads to blinding retinal degeneration.[37] Investigators have been able to develop an adenovirus construct of the missing gene, and have successfully inserted the gene into an animal model of gyrate atrophy. They have demonstrated that it successfully propagates in experimental animals.[25]

Researchers have also demonstrated the molecular genetic defects of OAT in gyrate atrophy patients.[38] For this purpose they constructed an eukaryotic expression vector (pcDHOAT), which contained the SV40 promoter and human OAT cDNA. They used OAT deficient Chinese hamster ovary (CHO) cells with negligible OAT activity. Fibroblasts from a gyrate atrophy patient (GA 35 cell) were used with negligible OAT mRNA and enzyme. Active enzyme was demonstrated in both cell types. The level of expression of human OAT was low in GA 35 cells in comparison to CHO cells. Despite the limited success, the ability to express active OAT in these OAT-deficient cells using an expression vector offers possibilities of gene therapy.

Lacorazza et al,[39] have successfully established an in vitro model to test the correction of OAT enzymatic deficiency in mammalian cells, using recombinant retroviruses. They have shown that the recombinant retrovirus transfers human OAT gene. Expression of the OAT gene in the transduced C9 deficient cells exceeded the OAT mRNA level and enzymatic activity of endogenous human fibroblasts.

Furthermore, there is strong evidence that the chorioretinal degeneration associated OAT deficiency is a consequence of hyperornithinemia. Therefore, development of metabolic system for clearing ornithine from the system is being pursued as a potential treatment. The skin is considered an attractive location for such a metabolic system because autologous cells can be safely and easily utilized.[40] This strategy uses an autologous keratinocyte graft modified to express high levels of OAT as an ornithine catabolizing skin enzyme.[41]

Nussenblatt and Csaky,[8] in another study adapted a strategy that used the skin of gyrate atrophy patients as the target for gene therapy with OAT to explore the possibility of direct gene insertion into the retinal pigment epithelium (RPE).

 Proliferative vitreoretinopathy (PVR)

PVR is the most common cause for failure of retinal reattachment surgery. It is characterized by pre- and/or subretinal membrane formation and contraction. It results in tractional retinal detachment with or without a rhegmatogenous component. Unscheduled proliferation and collagen synthesis of the RPE migrating through the retinal break cause membrane formation.[25] A key component of PVR is cell proliferation.

Wong et al[42] performed a study to examine whether the ribonucleotide-reductase-deficient HSV-I mutant hrR3 could effectively destroy proliferating RPE. It may prevent epiretinal membrane formation and PVR, while sparing non-dividing cells, such as neurons. They found that the hrR3 mutant strain of HSV-I could be used to infect and selectively kill actively proliferating rat RPE while sparing normal, non-replicating cells. Thus, this model may be used to explore therapies for PVR in humans.

Other investigators have demonstrated the application of gene therapy in animal model of PVR.[43],[44] They injected fibroblasts with or without retrovirus harbouring HSV thymidine kinase (HSVtk) gene. This makes the injected cells sensitive to gancyclovir, which was given after the injection. They found that PVR progression was inhibited by the retrovirally transferred HSVtk gene. Subsequent studies showed that an in vivo approach was also effective in preventing PVR.[43,44]

The feasibility of ocular gene therapy in a rabbit model of PVR has also been demonstrated using the HSVtk "suicide" gene. It was reported that although in vivo transduction efficiency was low, the strong "bystander effect" resulted in prominent killing of proliferating cells in the model leading to inhibition of disease. Thus in future, gene therapy may have the potential for replacement of defective gene products or introduction of new gene products into ocular cells.[45]

Though the pathogenesis of PVR is still unproven, growth factors are commonly believed to contribute to disease progression. While many growth factors have been implicated, platelet-derived growth factor is thought to be the strongest candidate for this process. Transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) have been implicated in PVR, because both TGF-β and HGF are mitogens for RPE cells.

Both are upregulated in PVR. These findings suggest that growth factors contribute to the development of PVR. Growth factors and their receptors may serve as targets for gene therapy based strategies to prevent PVR.[25]

 Experimental autoimmune uveitis (EAU)

EAU is a T-cell mediated autoimmune disease that targets the neural retina and serves as a model for human posterior uveitis. EAU can be induced against several retinal proteins in rats, mice, and subhuman primates. These include the S-antigen (a major protein in retinal photoreceptor cells), interphotoreceptor retinoid-binding protein (IRBP), rhodopsin and other antigens of retinal origin. There are many similarities between clinical uveitis and EAU. The latter differs in being self-limited, and needs adjuvant for disease induction. The experimental disease can be induced only in susceptible animal strains. Use of the EAU model has helped investigators understand the pathophysiology of the disease and evaluate disease-modifying agents, which could be applied to the clinic. Gene therapy may become a therapeutic strategy for EAU.[46]

Investigators have designed a strategy for EAU using the principle that immunoglobulins can serve as tolerogenic carriers for antigens, and B-cells can function as tolerogenic antigen presenting cells.[47] For this purpose a retroviral vector containing a major uveitogenic epitope of IRBP in frame with mouse IgG1 heavy chain was constructed. This construct was used to transduce peripheral B cells, which were infused into syngeneic recipients. A single infusion of transduced cells, 10 days before uveitogenic challenge, protected mice from clinical disease induced with the epitope or with the native IRBP protein. It was observed that the protected mice had reduced antigen-specific responses. These animals showed no evidence for a classic Th1/Th2 cytokine response shift or for generalized anergy. Protection was not transferable, arguing against a mechanism dependent on regulatory cells. Importantly, the treatment was protective when initiated 7 days after uveitogenic immunization or concurrently with adoptive transfer of primed uveitogenic T cells. This indicates that this form of gene therapy may induce epitope-specific protection not only in naive, but also in primed recipients. This observation suggests a protocol for the treatment of established autoimmunity.

 Corneal diseases

Diseases of the cornea are particularly amenable to gene therapy since vectors may be readily placed onto the corneal epithelium. Currently, insufficient gene transfer technologies and safety concerns prevent its broad application in humans.[48] However, a wide spectrum of applications can be exploited so that treatments for hereditary diseases, inflammatory disorders (herpes keratitis, autoimmune corneal melts), and rejection phenomena due to corneal transplant become possible. Preliminary data has provided some hope for the use of viral interleukin-10 in the treatment of experimental keratitis.[17]

 Other diseases

Retinal neovascularization occurs in proliferative diabetic retinopathy and in retinopathy of prematurity. New vessel growth in the choroid is the hallmark of ARMD, which affects the macula.[25] ARMD is a leading cause of blindness among the elderly.[49] It is likely that vascular endothelial growth factor (VEGF) plays an important role in the pathological angiogenesis. Upregulation of VEGF due to hypoxia in the retinal glial cells and/or the RPE is thought to be the strongest inducer for the growth of new vessels. Hence, VEGF and its receptor may serve as targets for gene therapy based treatments for this disease.[25] In addition, experimental studies are in progress to evaluate the potential of gene therapy in preventing conjunctival scanning in trabeculectomy failure.

Recently, it has been shown that naked plasmid DNA injected into the corneal stroma results in the efficient transfection of corneal keratocytes and epithelium. It has been shown that corneal neovascularization is promoted by biologically relevant levels of VEGF and inhibited its soluble receptor, respectively. It was reported that gene products were expressed within an hour of DNA expression, and persisted for at least 10 days. Thus, as protein-based therapeutics become more common, naked DNA gene therapy may prove useful in the treatment of corneal diseases.[50]

The use of non-viral vectors for gene delivery into the retina by liposome has also been reported. It used cytomegalovirus-promoted lac Z genes and non-histone nuclear protein. These were coated with the envelope of the Sendai virus to promote fusion with the cell membrane, and then injected (intravitreally and subretinally). Here lac Z expression appeared more transient than adenoviral vectors.[51] In addition, it was also reported that the Sendai virus liposome method could also be used as a non-viral gene therapy for the treatment of choroidal neovascularization.[52] The animal model of retinoblastoma provides the opportunity to study the process of malignant transformation in retinoblastoma and also promises to assist with development and testing of drugs to treat retinoblastoma.[53],[54]

Acland et al,[55] have used a recombinant AAV carrying RPE 65 to test the efficacy of gene therapy in a large animal model (dog) of Leber congenital amaurosis (severe retinal degeneration) which causes almost total blindness in infancy as a result of mutations in RPE 65. Their results demonstrate that visual function was restored in this large animal model of childhood blindness.

Bennett et al[56] had also tested the possibility of altering the course of retinal degeneration in mouse through subretinal injection of a recombinant replicative defective adenovirus. Subretinal injection resulted in delaying photoreceptor cell death by six weeks. This finding demonstrated cell rescue by in vivo gene transfer, supporting the feasibility of treating an inherited retinal degeneration by somatic gene therapy.

 Obstacles to successful human gene transfer

Gene therapy raises the prospects of treatment of diseases for which there has been no real hope of cure. However, inflated claims of the potential of gene therapy continue to raise unrealistic expectations. For gene therapy to work, the correct genes have to enter the appropriate cells, and operate for a prolonged period without any adverse effects. Serious problems remain at each stage in achieving these goals.[57]

Development of effective and safe ocular gene therapy requires an understanding of the genetic regulatory mechanisms. This understanding is needed to design therapeutic strategies that will lead to the expression of the desired gene into the appropriate cells. Otherwise, inappropriate and aberrant expression could lead to serious problems.[58]

In addition, not only is the correct gene product expected to be expressed in the appropriate cells, it must also be expressed at the optimal level. Too little expression could lead to a failure to correct the disorder, while too much expression could have an adverse effect. The potential danger of excessive expression even in the appropriate cells is demonstrated by the observation that transgenic mice expressing high levels of wild-type human rhodopsin develop retinal degeneration.[58,59]

Gene therapy research continues to be hampered by the difficulties of inserting genes into cells both by viral and non-viral vectors. Viruses may trigger an immune response rendering the newly inserted genetic material ineffective. Some retroviruses are relatively poor at invading non-dividing cells. Physical methods tend to be short lived, with gene expression lasting for a few days to weeks. Thus, the search for suitable vectors remains one of the biggest challenges for gene therapy.[22],[37] Lack of appropriate larger animal models has slowed the progress in clinical trials. In addition, the relatively small size of the eye limits the injection volume, and hence the gene delivery system must be highly effective.[59],[60]

Gene therapy often uses viruses to transport therapeutic genes into cells. These viruses can become widely dispersed in the body, and animal studies have shown that they can end up in gonads. A patient in a gene therapy trial had the virus used to transfer the gene show up in his semen.[61] This finding highlights the danger that gene therapy may inadvertently modify the genetic make-up of the off spring. It was not known initially whether the virus actually entered the sperm, as semen also contains other cell types such as white blood cells. However, the sperm tested negative for virus gene fragment in this particular instance.

Though many hurdles remain that must be overcome in order to use gene therapy routinely as a therapeutic approach in the clinic, these should be embraced as challenges that can be met with greater knowledge and research.

 Safety and ethics

Although gene therapy has been heralded as a success in medical science, it also carries the potential for abuse for commercial imperatives. Therefore, before human trials for gene therapy can commence for a given disease, extensive preclinical studies must be carried out in appropriate experimental models to assure the feasibility, safety and efficacy of the enterprise. For each disease or application, the risk/benefit ratio must be determined. Permission from regulatory agencies, institutional review boards and thorough informed consent must be made mandatory.[62]

Rapid development of gene therapy and the increasing tendency to apply them for certain human diseases has created a number of ethical problems. The first and foremost concern is direct intervention in the genome of germ line cells, which may change the genome of future generations. It is associated with a possibility of uncontrolled spreading of viral (particularly retroviral) constructs used in many gene therapy protocols.[63]

Ocular gene therapy seems poised to be amongst the successful applicants of this new approach to disease and pathology.[64],[65] However, the complications and risks are attributable because the patient may develop an immune response to the virus. Therefore, researchers must be well advised to build a strong preclinical case for safety and efficacy before proceeding to the clinic for any ocular applications. Hence, ocular gene therapy is a subject of greater oversight than all other specialties to protect patients as well as to ensure the progress of research.

 Future directions

Gene therapy holds the promise of curing ocular diseases, and improving the quality of life for millions who suffer from visual impairments. However, it is clear that the ideal way to introduce genes into the ocular tissues still needs to be well defined.

Several new biological treatment approaches have emerged which have shown some efficacy in animal models. They have to be tested for longterm effectiveness and safety before they can be investigated in humans. These include the use of growth factors to delay cell death, cell transplantation (i.e., replacing photoreceptor cells or RPE cells with new healthy ones) and gene therapy aimed at replacing the mutated genes with non-defective genes.

It is difficult to predict the key factors required for success of ocular gene therapy. Delivery of transgene to the target tissue, regulation of gene expression, host-immune responses to the vector and transgene product, and scaling up production of clinical grade viral based vectors are few of the tough challenges currently faced by researchers. Gene therapy with cytokines or their inhibitors is still in its infancy. However, with the increased understanding of the molecular genetics of eye disease together with the development of new approaches and new vectors for gene delivery make it likely that ocular gene therapy may become a reality.


Financial assistance from Indian Council of Medical Research towards research on the subject is gratefully acknowledged. Parul Tripathi is a Ph.D. student supported by SGPGI.


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