The mechanisms of interfering RNAs (iRNAs), in particular small interfering RNAs (siRNAs), have recently been investigated for their possible use in the treatment of various disorders. These RNAs have also shown promising results in the treatment of eye diseases, including glaucoma.
Glaucoma is a chronic degenerative eye disease that can lead to irreversible blindness and is characterised by progressive and irreversible damage to the optic nerve. Currently, the administration of topical anti-glaucoma drugs is the most common therapeutic strategy to reduce elevated intraocular pressure, which is the main risk factor.
Advances in iRNA research have opened up new treatment opportunities in the case of glaucoma through gene replacement and silencing, allowing defective genes to be replaced or corrected. In the ocular district, moreover, the peculiar anatomical structure is an advantage for local treatment: the eye is a confined compartment that serves as a good target for therapeutic use with siRNA.
Interfering RNA and siRNA: what they are
Since its discovery, iRNA has generally been used in research to suppress the in vitro and in vivo expression of various genes and is thus a valuable method for studying gene function. Soon, these characteristics were also taken into account for clinical application, and it was shown that siRNAs can be effectively used in therapy with a longer-lasting effect than existing pharmaceutical products.
siRNAs belong to a family of non-coding RNAs (ncRNAs), which regulate the function of genes, proteins and RNAs. They are small double-stranded RNAs (dsRNAs) with a length of 21 to 23 base pairs that can be exogenously administered and engineered to silence target genes. In the case of glaucoma, in particular, by suppressing the expression of specific genes, siRNAs are able to promote aqueous humour outflow, reduce its formation and act as neuroprotectors.
However, due to the high susceptibility to enzymatic hydrolysis, rapid removal from the circulatory system, off-target effects, low cellular uptake and possible immune response, clinical translation of iRNA has been more complicated than previously thought. Therefore, many vector systems have been designed to overcome the difficulties associated with siRNA delivery.
Gene vectors
The ideal gene vector should show minimal ectopic expression and effectively reach target tissues. In the case of chronic disease, gene expression should occur rapidly and continue throughout life, correcting the disease phenotype and preventing further degeneration. For siRNA, two types of vectors are mainly used: viral vectors and non-viral vectors.
- I viral vectors consist of genetically modified viruses as therapeutic transgenes and are the most widely used system due to their higher transfection efficiency in vivo. However, these systems have disadvantages related to limited payload capacity, mutagenesis and potential immunogenicity. The most commonly used viral vectors in ocular gene therapy are adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors and herpes simplex. The significant limitations associated with viral vectors have paved the way for research on non-viral vectors.
- I non-viral vectors have a lower risk of mutagenesis by insertion and are less immunogenic. They are easy to produce and can be mass-produced, are less toxic and do not cause any major ocular inflammatory response. In addition, they place no limits on the size of the gene they can carry. The main disadvantage of non-viral vectors is that they are less effective than viral vectors and rarely induce transgene expression at a therapeutic level. Based on the method of delivery, non-viral vectors are classified into chemical vectors and physical vectors. Chemical vectors include polymeric and cationic lipid-based nanosystems, while physical vectors include the use of gene-gun (a device that uses high-voltage electric current to deliver DNA to the target), electroporation, ultrasound and magnetofection.
siRNA delivery routes
The route of administration of siRNA to the eye is determined by the type of target cell and the characteristics of the vector. The simplest method is topical instillation, but intraocular bioavailability is very poor due to ocular barriers that limit the passage of xenobiotics such as nucleic acids. The periocular route involves the transfer of drugs/gens into the periocular region surrounding the eye and is considered the most efficient route of administration targeting the posterior segments of the eye (includes the subconjunctival, retrobulbar, peribulbar, sub-tenon and posterior iuxtascleral routes of administration). Intracameral injection involves the administration of the formulation in the anterior part of the eye and up to 100 μl volume can be injected by this route, but tends to have low efficacy due to the rapid turnover of aqueous humour. Subretinal injection occurs in the subretinal space that exists between the retinal photoreceptors and the retinal pigment epithelium layer. Suprachoroidal injections involve the administration of the formulation into the space between the sclera and the choroid, referred to as the suprachoroidal space.
Therapeutic targets in glaucoma
The main risk factor for glaucoma is an unusually high level of intraocular pressure, due to the eye's inability to adequately drain aqueous humour. The formation of this fluid and its evacuation are controlled by the sympathetic and parasympathetic nervous systems, respectively. When the balance between the formation and discharge of aqueous humour is disturbed, intraocular pressure increases. The most frequently used therapeutic strategies aim precisely at reducing intraocular pressure. Among the therapeutic targets investigated are the GTPase RohA, which regulates aqueous humour outflow, β2-adrenergic receptors, P2Y receptors, connective tissue growth factor (CTGF), activator of transcription factor-3 (ATF-3), Caspase 2 (which is over-expressed in glaucoma) and many others.
siRNA for the treatment of glaucoma
Currently, the most advanced clinical trials for the treatment of glaucoma with siRNA technology feature two promising molecules:
- SYL040012 is the siRNA molecule designed to silence β2-adrenergic receptors and is in phase II clinical trials. Blocking the β2-adrenergic receptor results in reduced aqueous humour formation and consequently reduced intraocular pressure. Undesirable effects are minimised as it is not absorbed by other cells or tissues in the body, with the exception of the ciliary body. The molecule is specific, effective and shows long-lasting results.
- QPI-1007 is a neuroprotectant targeting Caspase-2. It is currently in phase II clinical trials. The treatment is well tolerated, with low systemic toxicity.
In conclusion, siRNA-based therapy looks promising in all tissues, but it is particularly so in the ocular area as the specific anatomical characteristics of the eye offer an additional advantage. In the future, gene therapy for glaucoma could make it possible to provide a personalised, efficient and appropriate treatment of the disease for the individual patient and could improve the lives of many people already undergoing chronic treatment for the disease. To develop effective treatments, it will be crucial to have a greater understanding of the mutations that cause vision defects and to develop clinically efficient vectors.
Bibliography
Santoshi Naik et al. Small interfering RNAs (siRNAs) based gene silencing strategies for the treatment of glaucoma: Recent advances and future perspectives. Life Sci. 2021 Jan 1;264:118712. doi: 10.1016/j.lfs.2020.118712. Epub 2020 Nov 4.
Dr. Carmelo Chines
Direttore responsabile
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