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Not a ‘dry eye’ in the house

Ophthalmology has been relatively late to exploit the potential of microRNA and nanotechnology. However, now a ‘proof of concept’ study at the RCSI is showing their potential when used as combined therapies to treat dry-eye diseases.

Dr Sinead Connolly, Department of Molecular and Cellular Therapeutics, RCSI, will discuss the promise of microRNA and nanotechnologies for developing a new therapy for the rare Sjögren’s dry-eye disease, and the far more common non-Sjögren’s dry-eye disease, in a talk at the Irish College of Ophthalmologists Annual Conference 2017 in the Slieve Russell Hotel, Cavan, on 17 May. Her talk is titled ‘MicroRNA nanomedicines in Sjögren’s syndrome dry-eye disease’. 

Messengers

MicroRNAs were discovered in a C.elegans nematode in 1993, and in recent years there has been an explosion of interest in them. They are composed of about 20 of the four base pairs, adenine, cytosine, guanine and thymine, that make up DNA. They are essentially very short strands of RNA, the material that has the job of carrying instructions from DNA for making proteins.

Messenger RNA is one of a family of RNA molecules that carry instructions from the DNA to the ribosome for the manufacture of proteins. These messenger RNA molecules are specific, in that they bind to just one gene and thus act on one gene. MicroRNAs, in contrast, are non-specific and they can combine to form hundreds of potential messenger RNAs, which have the ability, as a result of these combinations, to activate or inactivate hundreds of genes.

MicroRNAs are, therefore, powerful cellular entities, which when over-expressed or under-expressed, can alter the whole phenotype of a cell and move it towards being, for example, a pro-inflammatory cell type. The wide-ranging ability that microRNAs have to influence cells has created a lot of interest among cancer biologists and immunologists, for example, who want to understand how microRNAs can become deranged in a variety of diseases.

Some diseases are the result of a single gene becoming deranged and it is possible in some cases to develop gene therapies to ‘knock-in’ or ‘knock-out’ that gene and cure the disease. However, with most diseases such as cancer, there isn’t just one gene to blame, but a whole range of genes that are turned on or turned off when the disease is expressed. The study of microRNAs can reveal what genes are turned off and on in a particular disease; thus, from a diagnostic point of view, they have potential as biomarkers of complex disease.

MicroRNAs are also highly relevant for developing new therapies for autoimmune disease where there are antibodies produced by the body attacking itself. The presence of a certain panel of antibodies could indicate whether a person has rheumatoid arthritis, lupus or Sjögren’s syndrome, a rare form of auto-immune disease. So there are potential benefits for ophthalmic disease that are autoimmune or inflammatory in origin.

The benefits of using microRNAs as a therapy, according to Dr Connolly, are that a wide-ranging effect can be created by targeting just one molecule. Then, from a drug delivery point of view, the drug only has to be delivered into the cell, not into the nucleus of the cell, which is a far easier job scientifically. “The field of microRNA research has exploded, certainly in other medical areas,” said Dr Connolly. “Ophthalmology has been a little slow to get interested in microRNAs, but certainly there is some work starting to come out,” she added.

Dry eye

Swedish ophthalmologist Henrik Sjögren described what is now called Sjögren’s syndrome in the 1930s. The syndrome is classified as rare and there are two forms (primary and secondary). The secondary form of the syndrome, which is associated with dry eyes and dry mouth due to gland disruption in the context of an existing rheumatological disease, is by far the most common form. 

Meanwhile, the incidence of primary Sjögren syndrome as an isolated condition is about one-in-1,000 and the ratio of females to males is about nine-to-one.

From work previously done in the group, said Dr Connolly, several microRNAs that were differently expressed were found on the ocular surface in Sjögren’s syndrome versus controls. The understanding is that microRNAs are in charge of whole cell processes, such as inflammation and cell death, so the way they are expressed in this case gives a clue as to why and how the syndrome occurs.

One microRNA that RCSI researchers were particularly interested in was an interferon-controlling gene, as it was known from previous work that patients with Sjögren’s syndrome have a high level of interferon circulating in their blood, but also a high level of interferon activity at the ocular surface.

“We were wondering whether it might be possible to target that microRNA to decrease interferon expression as a potential therapy, which is where I came in,” said Dr Connolly. Endogenously-produced microRNAs are also very stable, because they are released by cells as small packages, said Dr Connolly.

Strategy

The strategy was to investigate whether it was possible to package microRNAs so that the protein of interest could be targeted and to explore whether it could potentially be a new therapy for Sjögren’s syndrome, said Dr Connolly.

“The approach we came up with was using a nanoparticle formulation, which is based on a nanoparticle delivery system that is quite well used within our institution,” said Dr Connolly. “At the RCSI, there is a very active interest in respiratory research, especially cystic fibrosis, and the Department of Pharmacy had previously developed a nanoparticle system for delivery to lung mucosa.”

The properties that are good for lung delivery translate well into ocular systems, said Dr Connolly, so her research team tapped into their colleagues’ expertise in designing a nanoparticle delivery system. They spent a year-and-a-half doing this, she said, with lots of trial-and-error, before coming up with the best formulation possible in terms of its physical and chemical characteristics.

This process was challenging, given that nanoparticles are technically less than 1,000 micrometres, but to be useful they have to be even smaller — about 100 nanometres, which is absolutely tiny, said Dr Connolly. Changes are made in how much RNA and polymer are used in the formulation. They found the ideal formula after making many changes and testing how useful the formulations were.

The nanoparticle formula was placed on cells. Dr Connolly watched how this altered the microRNA and gene expression of the cells. It was important to get the experimental design right from the start as, unlike cancer, where hundreds of flasks of model cells can be grown quickly, the tissue samples were limited. The cells came from healthy volunteers and slightly altered conjunctival cells.

The scientists were able to ‘knock down’ the microRNA found to be over-expressed in the diseased cells, said Dr Connolly, and that led to an increase in the interferon regulation gene. This offers the prospect of a therapy that can knock-out the genetic triggers for Sjögren’s syndrome.  

Diagnosis

There are also important implications from this research for the diagnosis of Sjögren’s syndrome, which is a difficult condition to diagnose. Presently, there is no classification criteria, said Dr Connolly, either from the European League Against Rheumatism or the American College of Rheumatology. There are patients whom ophthalmologists may strongly suspect have primary Sjögren’s syndrome but do not meet the classification criteria and it can take anywhere between three-to-10 years, on average, to get a diagnosis.

Sjögren’s syndrome is also a difficult condition to treat. While it is rare, cases that present at the severe end of the dry-eye spectrum can take up a significant amount of a specialist’s time. For these reasons, ophthalmologists and patients will welcome any new therapies.

Non-Sjögren’s

Dr Connolly said there are also implications for the treatment of non-Sjögren’s dry-eye, as the same technology could be applied to this disease, which is a highly inflammatory condition in terms of its ocular surface biology. Dr Connolly said this is very exciting, as non-Sjögren’s dry-eye disease is a very common condition in ophthalmology.

“That is a huge potential market if you were looking at it from a pharma company point of view, with a huge patient population who aren’t adequately treated at present,” said Dr Connolly.

The potential also exists for the nanoparticle drug delivery system to be generalised for use in other conditions, said Dr Connolly. This would involve finding an RNA strand of interest, she said, packaging it, and then trying it out on a cell model of the disease that a researcher is interested in. A number of research groups at the RCSI are already doing this, she said.

“There are a variety of conditions, including things like osteomyelitis, renal disease, pulmonary disease, that are all using nanotechnology coupled with microRNA, so it is certainly an area of growing research interest,” concluded Dr Connolly.

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