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Editors Selection IGR 14-3

Response

Comment by Abbot Clark & Allan R. Shepard & J. Cameron Millar & Iok-hou Pang & Nasreen Jacobson & Wan-Heng Wang on:

26161 Adenoviral gene transfer of active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow facility in rodent eyes, Shepard AR; Millar JC; Pang IH et al., Investigative Ophthalmology and Visual Science, 2010; 51: 2067-2076

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Comment 1: The commenter points out the potential cargo size limitation of AAV vs adenoviral or lentiviral vectors that may be used to deliver TGF-β2 to the eye. Fortunately, the TGF-β2 coding sequence is 1245 base pairs and is well within the noted 2200 base pair AAV cargo limit.

Comment 2: The commenter questions how the delivery site of the adenoviral TGF-β2 may affect the interpretation of the data. Given that TGF-β2 is a secreted cytokine, the specific delivery site may be less of an issue as long as it is targeting cells which are bathed in the aqueous humor of the anterior chamber, as is indeed the case with similarly injected eyes with Ad.GFP (as noted in the Discussion section of the paper). Targeting specific subtypes of tissues following intraocular injection of a given virus may be obtained by the use of tissue-specific promoters, inducible promoters, or selected virus/virus serotypes.

Comment 3: The commenter wonders how the precise location of TGF-β2 expression in the anterior segment, e.g., engineering to affect cells upstream of the TM, may determine its effects. This may be obtainable by the use of a ciliary body/iris specific gene promoter such as OPTC in place of CMV on future viruses.

 Comment 4: The commenter is concerned with the inflammatory effects of the adenovirus. As noted, we have controlled for the inflammatory effect alone as being responsible for the IOP elevation; however, we agree that inflammation plus TGF-β2 overexpression may confound the situation. Additional studies, testing the effects of (1) locally or systemically delivered NSAIDs or the suggested TNFalpha inhibitor thalidomide, or (2) the effect of alternative viral delivery vectors on Ad.TGF-β2-induced ocular hypertension will be instructive. A larger eye model, e.g., rabbits, cats, non-human primates, as well as the other steps noted above may allow for more directed delivery and expression of the virus. We did show in the paper (Fig. 5) that aqueous humor levels in the mouse Ad.TGF-β2-injected eyes were elevated, but not supraphysiologically compared to human aqueous humor TGF-β2 levels.

Comment 5: Regarding the duration of in-vitro expression of Ad.TGF-β2, correlating in-vitro with in-vivo duration of expression data is challenging at best. Experiments towards this goal may be confounded by the use of low or serum-free (serum contains TGF-β) culture conditions and long-term health of the transduced cells in this environment. Measuring TGF-β2 levels over the course of the in-vivo study may be more relevant, but also a much larger undertaking. The question of whether the ciliary epithelium and aqueous production is affected by the adenoviral transductions is a good one and one which should be addressed. However, in our experience with Ad.GFP, transduction of the ciliary epithelium is a minor component and any effects of the TGF-β2 on aqueous inflow would likely be indirect. A longer lasting rise in IOP due to the mechanical changes in the outflow facility is suggested by the commenter. However, because the ECM content is dynamic, it is not unreasonable that once the selective pressure from the adenoviral TGF-β2 is removed, that the ECM renormalizes, which may provide hope for the clinical setting. Additional work would be needed to prove this point. Uveoscleral outflow can, at present, only be assessed indirectly in the rodent. However our data demonstrate that the reduction in conventional outflow can account for the increase in IOP with the Ad.TGF-β2 vector-injected eyes (as noted in the Results section). The use of anti-CD40L antibodies to prolong the Ad.TGF-β2 effects in the mice is a possibility, but we agree with the commenter, that other vectors with minimal host responses may be the better approach. We feel that the long-term effects of a physiologically relevant level of TGF-β2 may not necessarily prove an obstacle to developing an animal model of glaucoma and that inappropriate targeting of the TGF-β2 virus, e.g., lens-selective promoter, would be more of a concern. With the approaches highlighted above, we look forward to improving upon our model and developing the first disease-based full-spectrum animal model of glaucoma.

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