EDITORIAL

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AVANCES EN EL TRATAMIENTO DE LA RETINOPATÍA
PROLIFERATIVA MEDIANTE TERAPIA GÉNICA

ADVANCES IN A GENE THERAPY-BASED APPROACH TO TREAT
PROLIFERATIVE VITREORETINOPATHY

ANDRIUS KAZLAUSKAS Ph. D¹

Proliferative vitreoretinopathy (PVR) is the major cause for failure of retinal reattachment surgery for rhegmatogenous retinal detachment. While its occurrence is relatively low (approximately 10%) PVR remains a difficult disease to treat.

There are several hypothesis regarding the development and progression of the disease. Following the formation of a retinal break, retinal pigment epithelial (RPE) cells are dispersed into the vitreous. The proliferation of these cells and the deposition of extracellular matrix proteins contribute to the development of an epiretinal membrane. This membrane, which adheres to the inner and/or outer surface of the retina, grows and eventually contracts, causing the retinal detachment. The cells found in the epiretinal membrane are primarily RPE cells or fibroblast-like cells (which may be dedifferentiated RPEs), some glial cells, and to a much lesser extent macrophages (Baudouin et al., 1990; Vinores et al., 1990). The other obvious component of the epiretinal membrane is extracellular matrix, which is composed of collagens and fibronectin (Jerdan et al., 1989).

There has been a large effort directed toward better understanding the cell biology of the cells within the epiretinal membrane. The cells in this membrane produce a number of different growth factors, including platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) (Campochiaro et al., 1989; Lashkari et al., 1999; Schneeberger et al., 1997). The very cells that are producing the growth factors often express the receptors to respond to them. For instance, RPE and glial cells express the receptors for PDGF, HGF and/or VEGF (Campochiaro et al., 1989; Guerrin et al., 1995; Lashkari et al., 1999; Leschey et al., 1990). Finally, growth factors such as PDGF promotes the synthesis of extracellular matrix proteins, proliferation of cultured RPE cells (Campochiaro et al., 1989) and their contraction in collagen gels (Choudhury et al., 1997). Thus growth factors are capable of driving the cellular responses that are required for both the growth and contraction of epiretinal membranes.

Gene therapy has enormous potential as an approach to treat and cure diseases, and it is likely to be the way many diseases are managed in the future. Current attempts to utilize gene therapy have revealed that the success of this approach requires at least the following three elements. 1) an efficient gene delivery system; 2) expression of the introduced gene must persist long enough to have an effect; 3) the host-immune reaction must be minimized (Verma and Somia, 1997). The eye is a particularly attractive organ for gene therapy because it is accessible, anatomically isolated and immune privileged.

For the last 5 years we have been working on a gene therapy-based approach to prevent PVR. The first step was to identify a gene that was required for PVR. Based on the existing literature we hypothesized that PDGF or the receptor for PDGF was such a gene. This hypothesis predicted that cells that could not respond to PDGF would be unable to induce PVR. While RPE cells are the most relevant cell line for PVR, they naturally express the receptors for PDGF and this made it difficult to systematically test the requirement for PDGF receptors (PDGFRs). Instead we used a mouse embryo fibroblast cell line that was derived from embryos that lacked both PDGFR genes (Andrews et al., 1999). These cells failed to induce PVR when they were coinjected with platelet-rich plasma into rabbits that had previously undergone gas vitrectomy (Andrews et al., 1999). Expressing the PDGF a receptor (aPDGFR) in these cells greatly improved their PVR potential, whereas cells expressing the related PDGF b receptor (bPDGFR) were unable to induce the disease (Andrews et al., 1999). These findings identified the aPDGFR as a gene that was essential for PVR in the rabbit model of the disease.

The second step to develop a gene therapy-based approach to prevent PVR was to design a strategy to prevent cells from responding to PDGF. To this end we created and characterized a series of dominant negative PDGFRs, which were designed to interfere with activation of the wild type PDGFR and thereby render the cells unresponsive to PDGF. Five different dominant negative receptors were created and found to efficiently block PDGF-dependent responses in cultured cells. We then tested the capacity of these dominant negative PDGFRs to prevent PVR. Instead of using mouse embryo fibroblasts to induce the disease (as described above) we used primary rabbit conjunctival fibroblasts, which naturally express the PDGFR and have a very high PVR potential. The dominant negative PDGFRs were expressed in these cells and the resulting cells were tested for their ability to induce PVR. All 5 of the dominant negative PDGFRs attenuated PVR, and the truncated dominant negative was the most effective one (Ikuno et al., 2000). These ex vivo studies reinforced the importance of PDGF in PVR and identified the best dominant negative PDGFR for blocking the disease.

We extended these ex vivo studies with an in vivo series of experiments. To better approximate the clinical setting we wanted to administer the dominant negative PDGFR to the cells after they had been injected into the vitreous cavity. This differs from the ex vivo setting in two ways. In the ex vivo setting the cells that were injected in the animal were pre-selected for expression of the dominant negative PDGFRs, and the gene was introduced under optimal conditions in tissue culture. In the in vivo setting there would be no selection step and the gene delivery would occur in the vitreous cavity of the eye. We compared viral expression systems and found that retroviruses were both an efficient and selective vehicle for expressing genes in rabbit conjunctival cells that had been injected into the vitreous cavity. Next we tested if delivering the truncated dominant negative PDGFR via a retrovirus impacted PVR, which was induced by a prior injection of conjunctival fibroblasts. Our results indicated that the answer was yes. Although PVR was not eliminated in these animals, the truncated dominant negative PDGFR caused a significant attenuation of the disease (Ikuno and Kazlauskas, 2002).

A gene therapy-based approach using a dominant negative aPDGFR delivered with a retrovirus is effective in attenuating PVR in a rabbit model of the disease. It should be interesting to test this approach in other PVR models and of course in humans when it is safe and ethically sound to do so. Finally, we plan to investigate the molecular basis of the difference in the PVR potential between the aPDGFR and bPDGFR. It is our hope that these efforts will increase our understanding of the disease, which will guide efforts to develop approaches to treat and/or prevent PVR.

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¹ Schepens Eve Research Institute/Harvard Medical School. USA
E-mail: kazlauskas@vision.eri.harvard.edu

 

BIBLIOGRAFÍA

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