Introduction
Gene therapy has been a logical approach for retinal diseases caused by mutation and resulting phenotypes. The recent work in gene therapy has focused on optimizing the design of the vectors to promote optimized expression and phenotypic rescue after intraocular delivery. Despite the success of viral gene therapy for treating retinal diseases, there remains significant room for further improvements. Even though the newer viral vectors have been non-integrating and self-inhibiting, a continuous need for refinement and development of gene therapy is still needed for the vectors of the eye. Hence, significant research efforts are directed towards developing a non-viral delivery system.
What Is Gene Therapy and Its Types?
Ocular gene therapy is a potentially disruptive technology for inherited and acquired retinal conditions. The term “gene therapy” encompasses strategies for replacing non-functional genes with normal copies of inherited retinal diseases. The mainstay of this system involves the intraocular delivery of viral and non-viral vectors, which can carry the therapeutic gene into the nuclei of the target cell. The efficacy is determined by the vector choice and the administration route for establishing a safe platform. Recent advances have broadened the options available as well.
The type of gene therapy includes:
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Gene Augmentation: This is the addition of a gene to a cell.
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Gene Editing: Revision of the existing genetic code.
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Gene Inactivation: Silencing a gene, mostly the negative one, which is dominant.
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Selective Toxicity: Recognition of cancer cells.
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RNA Therapeutics: Targeting the RNA instead of the DNA within a cell.
Ocular gene therapy mostly involves the augmentation of genes. The majority of gene therapy targets autosomal recessive ocular diseases.
What Is the Need for Genetic Testing in Ophthalmology?
Genetic testing has been recommended whenever there is a retinal dystrophy, as it might be associated with genetic mutations. Genetic testing can be confirmatory for IRDs; it can provide an exact diagnosis and management strategy, including risk assessment and family counseling. The gene testing results are not binary and present a ranking system for each identified mutation based on the standards. The pathogenicity is determined based on various factors, like the effect on the coding of genes, the structure and function of the protein, and the associated variants of the disease within a population.
What Are the Categories of Gene Therapy?
Gene therapy is mainly divided into three categories.
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Firstly, gene replacement therapy is available for diseases associated with loss of function mutation, a common mutation in retinal-specific genes.
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The second one involves using knockdown technology, which eradicates the mutant alleles associated with gain of function mutation.
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The third group aims to design genetic treatment for neurodegenerative disorders like (glaucoma and age-related macular degeneration).
What Are the Different Forms of Non-viral Gene Delivery?
There are various forms of nonviral gene therapy:
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Naked DNA: It is the most basic form of nonviral gene therapy administered by almost all delivery systems. It is typically not taken up into the cells.
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Liposomes: These are being extensively studied, that is, the cationic lipids. Here, the DNA-encapsulating gene is transferred to the eyes.
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Compacted DNA Nanoparticles: These vectors offer a large payload, there is no generation of any toxic response, and due to their small sizes, they offer an efficient cell uptake. Mostly, they are 10 to 400 nm in diameter. There is a second approach of nanoparticle condensation of a single molecule of linear or plasmid DNA or RNA to a diameter of 8 to 20 nm. This particular technology of nanoparticles can drive a high level of gene expression throughout the eyes.
What Are the Methods of Non-viral Gene Delivery?
Various methods have been developed to deliver non-viral genes in the past two decades. The nonviral delivery systems are still under investigation. According to the clinical needs, they have the potential for titration and repeated dosing, according to the clinical needs. Single DNA molecules have also been explored. This nonviral gene transfer would ensure a lesser risk of immune response in patients with pre-existing viral immunity; a very large transfer of genes can happen, which is beyond the capacity of the viral gene delivery system.
The delivery route of the nonviral gene therapy vector remains crucial for the treatment and leads to the highest transfection rate for the targeted cell type, which has the least risk for severe adverse effects. The topical application offers the least noninvasive delivery and is mostly used for anterior segment diseases like glaucoma. Intravenous injections offer yet another way by which compounds can be administered directly into the bloodstream. Periocular injection is another type of drug being administered apart from subretinal and intravitreal injections.
Why Nonviral Gene Therapy Is Preferred?
The viral vectors offer immunological reactions. The nonviral therapy promises to be less immunogenic than the viral ones. The gene size is not limited; this allows the delivery of the entire genomic DNA fragment, including the gene regulatory elements and intronic sequences, to increase expression efficiency. The production charges of nonviral gene therapy are a lot less compared to that of viral ones. Moreover, the nonviral delivery system gets classified as drugs rather than biologics, cutting costs and administrative efforts.
What Are the Challenges of Non-viral Gene Therapy?
As known, the subretinal injection is usually performed to target the photoreceptor, but the same for the naked plasmids by no means results in significant gene expression. A DNA plasmid may not have the vehicle to overcome the natural cellular barrier to viral infections. It may be exposed to intracellular nucleases, leading to rapid degradation of the naked DNA. Several obstacles must be overcome before the gene delivery results in protein expression. Also, the agents and procedures used to assist and facilitate gene delivery must not be toxic to the retina.
Additionally, another problem remains: even if the DNA gets successfully delivered to the nucleus, the gene would persist in the nucleus and continuously express the proteins. Therefore, there is a need to develop DNA delivery methods that allow easy passage through the cell membrane and ideally help to overcome further barriers and silencing. Many such ways have been developed in vivo and in vitro, and DNA delivery to the outer retina has also been tried.
What Are the Optimization Techniques for Non-viral Gene Therapy?
There are various ways to improve gene delivery efficiency, independent of the methods used for nonviral gene therapy. The most promising techniques include:
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Vector Integration: It allows for achieving persistent expression.
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Scaffold Matrix Attachment: It is suggested as it helps overcome gene silencing for non-viral retinal gene therapy. These attachment regions are thought to isolate the active from the inactive chromatin and protect the DNA from methylation.
Conclusion:
Non-viral gene therapy has several advantages, but its success remains limited. It remains to target the photoreceptor cells and achieve a high and persistent transgene expression without causing any harm to the neuronal tissues by using physical and chemical transfection methods. Thus, to better estimate non-viral gene therapy, it should be tested against the viral vectors in the future.
