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Anterior segment 
gene therapy

Achievements in the laboratory create optimism for clinical translation.

Cheryl Guttman Krader

Posted: Tuesday, October 1, 2019

Gene therapy has been successful in preclinical studies as intervention for various conditions affecting the anterior segment, and the results hold promise for clinical application in the future, reported Alexander V Ljubimov PhD at the 2019 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO) in Vancouver, Canada.
Providing an overview of gene therapy for anterior segment disease, Dr Ljubimov described research using various vectors and focusing on a growing number of molecular targets. He noted that current trends include the prevalence of topical gene administration and increasing use of nanocarriers for gene delivery. Gene-editing techniques for inherited diseases are also emerging as a gene therapy variant that is beginning to be applied to corneal dystrophies.
“There is a race to translate these systems to the clinic and to obtain FDA approval,” said Dr Ljubimov, Director, Eye Program Board of Governors Regenerative Medicine Institute, and Professor of Biomedical Sciences and Neurosurgery, Cedars-Sinai Medical Centre, University of California Los Angeles School of Medicine, Los Angeles, USA.
The ease of access to target tissues and the availability of a variety of diagnostic tools that can be used for monitoring outcomes make gene therapy an attractive therapeutic option for diseases affecting anterior segment structures. Currently, progress is being made with gene therapy for diseases affecting the cornea, conjunctiva, lacrimal glands and trabecular meshwork.
“Gene therapy has been successfully tested in various model systems and in animals using various viruses for delivery as well as using plasmids, microRNA, self-delivery siRNA and nanoconstructs. Administration routes have included topical, intrastromal, subconjunctival and anterior chamber injections. Of course, non-invasive delivery with topical administration represents the ideal scenario,” said Dr Ljubimov.

Corneal gene therapy

Dr Ljubimov’s research focuses on corneal gene therapy and he devoted a portion of his talk to describing the work being done in his laboratory to address corneal epithelial wound healing problems in patients with diabetes.
Based on the identification of various molecular alterations in diabetic corneas, Dr Ljubimov and colleagues developed adenovirus-based gene therapy approaches targeting those abnormalities and tested them in both cultured human diabetic limbal epithelial cells and in a human diabetic corneal organ culture system. He reported the best results were achieved with a triple combination approach that was designed to silence matrix metalloproteinase-10 and cathepsin F and upregulate c-met proto-oncogene.
“The combination gene therapy essentially normalised epithelial healing time as well as the expression of several putative epithelial stem cell markers,” he said.
Although the expression of transgenes was not negatively impacted using the combination approach, toxicity was encountered in stem cell-enriched cultures. To overcome that problem, an alternative treatment was designed using a non-toxic nanopolymer that had covalently attached antisense oligonucleotides inhibiting cathepsin F and microRNA-409-3p, which suppresses c-met expression.
Results of experiments using the latter approach showed that wound healing was accelerated in both the cultured cell system and whole diabetic corneas. In addition, restoration of the expression of putative stem cell markers – keratin 15, keratin 17 and ABCG2 transporter – was documented with immunostaining and Western blots. “Now we hope to expand the approach to target additional markers and perhaps explore combination therapy,” Dr Ljubimov said.
Gene therapy with delivery of the β-glucuronidase gene has been successful for ameliorating mucopolysaccharidosis type VII. Researchers have also used decorin, Smad7, BMP7 and c-met genes, antisense to opioid growth factor receptor gene, and an inhibitor of microRNA-146a to alleviate scarring and burn injury and to enhance wound healing.
Gene therapy is also being investigated for its potential to improve corneal graft survival with approaches that include silencing or upregulating certain interleukin genes or transfer of the Bcl-xL gene to reduce apoptosis that is associated with graft failure.
Herpes simplex virus (HSV) keratitis is another target for corneal gene therapy research. Studies have been done targeting HSV glycoprotein genes, inflammatory mediators and degradation of the HSV genome.
In addition, pathologic corneal neovascularisation has also been successfully reduced through transduction of s-Flt-1, Flt23K, pigment epithelium-derived factor, endostatin genes and microRNA-204.

Other anterior segment targets

Gene therapy approaches that have been successful in the laboratory for counteracting conjunctival scarring include those that increased expression of Smad7, PPARγ and the dominant negative mutant for p38MAP kinase. Gene therapy directed at the lacrimal gland is being studied as a possible treatment for dry eye disease. Alleviation of dry eye symptoms has been demonstrated using adenoviral-based gene therapy targeting TNF-α inhibitor, and interleukin-10 gene therapy was shown to improve signs of Sjögren’s syndrome. In addition, increased tear production has been achieved using nanoparticle delivery of the MUC5AC gene.
Gene therapy for glaucoma is focusing on increasing outflow facility through the trabecular meshwork, and research in this area includes attempts to increase expression of cyclooxygenase-2, prostaglandin F synthase and matrix metalloproteinase-3. In addition, CRISPR/Cas9-based gene editing in a mouse model of myocilin-associated glaucoma was shown to knockdown expression of the mutant myocilin gene and reduce IOP.

Ljubimov@csmc.edu: Ljubimov@csmc.edu