Glaucoma Opinion IGR 12-1

Gene Therapy in Glaucoma

Terete Borrás, Chapel Hill, NC, USA

In theory, gene therapy appears as it could be the panacea of all treatments. Genes make proteins and proteins become active enzymes. With the availability of a complete sequenced human genome, one could choose (nowadays, buy) any fully functional gene. For genetic diseases, where the genetic defect is known, one could replace the missing good gene, or silence the deleterious gene product. For non-genetic, acquired syndromes, one could use the gene as a drug, and target a mechanism relevant to the disease. Knowledge on gene regulation would allow one to direct the therapeutic gene to the correct tissue and to turn it on/off in response to external signals (e.g., a glaucomatous insult). A single gene delivery could become, like a vaccine, one long-term treatment. Such treatment would be more specific, and far less toxic than conventional drugs.

Using genes as drugs that modulate significant pathways is quite feasible, and within our grasp. Gene therapy appears as it could be the panacea of all treatments

In reality it is neither that simple, nor straightforward. Each small step requires tremendous work. Key parameters for successful gene therapy, such as delivery vectors, selection of genes and their regulatory elements, need to be tailored and rigorously engineered to achieve the desired effect with minimal toxicity. Appropriate animal models are a must.

Because glaucoma is a complex disease and many different genes are (and would be) linked to the disorder, the route of developing a gene therapy product to repair each glaucoma-associated gene is not yet practical. However, the approach of using genes as drugs that modulate significant pathways is quite feasible, and within our grasp.

The route of developing a gene therapy product to repair each glaucoma-associated gene is not yet practical

Vectors to deliver genes to cells are constantly being improved. Because viruses penetrate our cells easily, replication-deficient recombookbinant viral vectors are favored over naked DNAs. Adenoviruses (Ad), Adeno-associated viruses (AAV) and lentiviruses (HIV), together with their various serotypes, have different cell tropisms, packaging capacities and immunological properties. For the eye, AAV is emerging as the preferred vector.1,2 AAV's main limitation, its low capacity for large genes, is currently being challenged.3

For gene therapy of glaucoma, the focus has been primarily in lowering elevated IOP and in neuroprotection of retinal ganglion cells (RGC).

Trabecular meshwork (TM)

Gene delivery to the TM has been accomplished in vitro, organ culture and living animals. The first vector used, Ad vector, proved to be a very efficient transgene carrier but had a short life (4-5 weeks). An optimized AAV (self-complementary AAV, scAAV) did much better, and after a single intracameral injection expressed the transgene in the monkeys' TM for 2.3 years.2 No clinical or cellular signs of inflammatory response were observed.2 A feline lentiviral vector (FIV) showed transgene expression when harvested ~1 year after injection.4

Regarding selection of gene therapy TM genes, both classical and new knowledge have yielded good candidates. Overexpression of caldesmon and Rho pathway genes, which disrupt the cytoskeleton, increased outflow facilities in perfused organ cutures.4 Delivery of the cyclooxygenase-2 gene (COX-2), involved in prostaglandin biosynthesis, combined with its receptor PGF , lowered IOP in cats for five months.5 Many additional candidate genes have been identified by microarray chip analysis6 and a few are in the pipeline (MMP1, angiopoietin-like7, collagen-elastin crosslinking enzymes). Inducible expression has also been achieved for steroid glaucoma. A vector carrying the MMP1 gene under the control of glucocorticoid- response elements (GRE), reduced elevated IOP induced by triamcinolone and prednisolone in sheep.7,8

Retinal ganglion cells (RGC)

For the RGC, gene therapy efforts are being directed to prevent apoptotic RGC death. In contrast to Ad and lentiviruses, intravitreal delivery of AAV vectors provided efficient, long-term transduction to RGC cells.1 This transduction can be inducible.9 A tetracycline-regulated GFP, delivered by a tetracycline-transactivator AAV2 vector, was turned off after oral administration of doxycycline. The on/off switch went on for two cycles over six months.9

Transgenes used so far to prevent RGC death comprise mainly apoptosis inhibitors, neuroprotrophic factors and oxidative stress regulators, all delivered by AAV2 vectors. Thus, the inhibitor of apoptotic cytochrome C release, Bcl-xL, controlled by the synapsin1 promoter, protected 46% of transduced RGC in rats at eight weeks post-axotomy. 10 The neurotrophins BDNF and CNTF showed RGC protection in two rat glaucoma models.11,12 And overexpression of free radical scavengers, such thioredoxins, also preserved RGCs.13 An example of other genes targeting newly identified protective pathways could include calcineurin, amyloid- , γ-synuclein and coenzyme Q10.

Concluding thoughts

Slowly, but surely, gene therapy for glaucoma is moving forward. Although optimization continues, we now have viral vectors that elicit minimal immune response and achieve long-term gene transfer to the anterior and posterior segments. We also have many candidate genes that can target relevant mechanisms.

In the not-so-distant future gene therapy will become a standard treatment for glaucoma
The next big challenge lies in regulating transgene expression, in identifying cell-specific promoters and inducible DNA elements, which will express the gene where we want, and when is needed. And we need to focus on a few good genes. Not small, but achievable goals. I have no doubt that in the not-so-distant future gene therapy will become a standard treatment for glaucoma. I have no doubt that in the not-so-distant future gene therapy will become a standard treatment for glaucoma.


  1. Harvey AR, Kamphuis W, Eggers R, et al. Intravitreal injection of adenoassociated viral vectors results in the transduction of different types of retinal neurons in neonatal and adult rats: a comparison with lentiviral vectors. Mol Cell Neurosci 2002; 21: 141-157.
  2. Buie LK, Rasmussen CA, Porterfield EC, et al. Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys. IOVS 2010; 51: 236-248.
  3. Hirsch ML, Agbandje-McKenna M, Samulski RJ. Little vector, big gene transduction: fragmented genome reassembly of adeno-associated virus. Mol Ther 2010; 18: 6-8.
  4. Liu X, Rasmussen CA, Gabelt BT, Brandt CR, Kaufman PL. Gene therapy targeting glaucoma: where are we? Surv Ophthalmol 2009; 54: 472-486.
  5. Barraza RA, McLaren JW, Poeschla EM. Prostaglandin pathway gene therapy for sustained reduction of intraocular pressure. Mol Ther 2010; 18: 491-501.
  6. Comes N, Borrás T. Individual molecular response to elevated intraocular pressure in perfused postmortem human eyes. Physiol Genomics 2009; 38: 205-225.
  7. Gerometta R, Spiga MG, Borrás T, Candia OA. Treatment of Sheep Steroidinduced Ocular Hypertensionwith a Glucocorticoid-inducible MMP1 Gene Therapy Virus. IOVS 2010; 51: 3042-3048
  8. Spiga MG, Borrás T. Development of a Gene Therapy Virus with a Glucocorticoid- inducible MMP1for the Treatment of Steroid Glaucoma. IOVS 2010; 51: 3029-3041
  9. Folliot S, Briot D, Conrath H, et al. Sustained tetracycline-regulated transgene expression in vivo in rat retinal ganglion cells using a single type 2 adenoassociated viral vector. J Gene Med 2003; 5: 493-501.
  10. Malik JM, Shevtsova Z, Bahr M, Kugler S. Long-term in vivo inhibition of CNS neurodegeneration by Bcl-XL gene transfer. Mol Ther 2005; 11: 373-381.
  11. Cheng L, Sapieha P, Kittlerova P, Hauswirth WW, Di Polo A. TrkB gene transfer protects retinal ganglion cells from axotomy-induced death in vivo. J Neurosci 2002; 22: 3977-3986.
  12. Pease ME, Zack DJ, Berlinicke C, et al. Effect of CNTF on retinal ganglion cell survival in experimental glaucoma. IOVS 2009; 50: 2194-2200. 13. Munemasa Y, Ahn JH, Kwong JM, Caprioli J, Piri N. Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma. Gene Ther 2009; 16: 17-25.

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