|Molecular Vision 2005;
Received 17 March 2005 | Accepted 1 June 2005 | Published 21 June 2005
Lentiviral mediated gene delivery to the anterior chamber of rodent eyes
Paloma B. Liton,1
Beth S. Chamblin,2
John K. Wakefield,2
David L. Epstein,1
1Department of Ophthalmology, Duke University, Durham, NC; 2Tranzyme, Inc., Research Triangle Park, NC
Correspondence to: Pratap Challa, MD, Box 3802 DUMC, Duke University Eye Center, Durham, NC, 27710; Phone: (919) 684-3283; FAX: (919) 681-8267; email: Chall001@mc.duke.edu
Purpose: To study the in vivo efficiency of lentiviral vectors in delivering genes to the trabecular meshwork (TM) of rodent eyes.
Methods: Lentiviral vectors were constructed using the elongation factor 1 alpha (EF-1α) promoter driving expression of the green fluorescent protein (GFP) gene. The viral construct was injected intracamerally through the peripheral cornea into the anterior chamber of live rodent eyes. Several variables were evaluated to determine the optimal conditions for TM cell transduction. These parameters included viral concentration, injection volume, needle rotation, and the modulation of anterior chamber current convections. Changes in intraocular pressures (IOPs) were monitored using a Tonopen XL. Signs of inflammation and corneal neovascularization were evaluated by slit lamp observation. Three weeks after injection, the eyes were enucleated and analyzed for GFP expression and distribution.
Results: A single intracameral viral dose between 107 and 108 pfu produced a high and evenly distributed expression of GFP in the TM and corneal endothelial cells. The cornea remained clear and no signs of inflammation were present during the course of the experiment. Moreover, no significant changes in IOPs were observed.
Conclusions: A high transduction efficiency of TM and corneal endothelial cells can be effectively obtained after a single dose of recombinant lentivirus. The EF-1α promoter induces high expression of the reporter gene and is a reliable alternative to the CMV promoter when stable, long term expression is desired.
Primary open angle glaucoma (POAG) is a leading cause of irreversible blindness and is characterized by progressive optic nerve degeneration that eventually produces visual field loss. Elevated intraocular pressure (IOP) is a major risk factor for POAG and appears to result from increased trabecular meshwork (TM) outflow resistance [1-3]. In vivo gene transfer is a powerful technique with broad applications for both experimental studies and future gene therapy. In general, the eye has a number of advantages as a gene transfer target. It is readily accessible, relatively immunoprivileged, and has a wide range of potentially transducible tissues. Moreover, the majority of aqueous humor leaves the anterior chamber through the TM filter system making this tissue a natural target for gene delivery.
Several viral systems have been reported for gene delivery to the eye; baculovirus , herpes simplex virus (HSV) , adenovirus [6-8], adeno-associated virus (AAV) [9,10], and, more recently, lentivirus [11,12]. Most of these experiments have targeted retinal tissues for transduction. Furthermore, adenovirus, AAV, HSV, and lentivirus have been used for in vivo gene transfer to the anterior chamber in several animal models. Adenovirus has been used for anterior chamber transfection in rats , mice , monkeys , and rabbits . AAV has been used in rats  and rabbits  while HSV has been used in rabbits  and lentivirus in felines . Each of these viral vector systems has different biological properties.
Lentiviral vectors have several advantages and disadvantages when compared to other frequently used viral systems such as adenovirus, AAV, and HSV. Compared to adenovirus and AAV, lentiviruses, in general, have more stable and long term expression once they integrate into the host genome. They can infect both dividing and nondividing cells and integrate into host genomes with low immunogenicity and high transduction efficiency [20,21]. The main disadvantages in using lentiviruses as gene delivery vectors are that large scale production is relatively costly and time consuming, and there are safety concerns in generating replication competent viruses.
AAV shares many of the advantages of lentiviruses plus the added benefit that it is not associated with any known pathology in humans. However, limitations of AAV include difficulty in preparing high viral titers , and payloads limited to 4 kb as opposed to lentiviruses and adenoviruses which can carry up to 8 kb . Adenoviral vectors can be easily purified to high titers, demonstrate low pathogenicity, and provide high transduction efficiency . However, adenoviral gene transfer results in short term transgene expression. More importantly, they elicit cellular and humoral immune responses [20,24] that can interfere with the interpretation of experimental results. The use of HSV for gene transfer in vivo has also been shown to have an associated inflammatory immune response [25-27]. Therefore, the use of lentiviral vectors could potentially represent a method of choice for long term and stable transgene expression.
Transduction of TM cells using lentiviruses has been recently reported in felines . However, rodents constitute a more practical animal model than cats for the study of the physiology of the outflow pathway. They are inexpensive, easy to handle, and have nearly complete genome sequences. Furthermore, their outflow pathway is similar to that of the human eye. However, a drawback of using rodents for aqueous outflow studies is that their small anterior segment size makes gene delivery a technically challenging task. Therefore, we investigated the feasibility of gene transfer to the anterior segment of living rodent eyes using lentiviral vectors. We also optimized the experimental conditions to achieve transgene expression distribution throughout the entire circumference of the TM while minimizing damage to the corneal endothelium.
All animal procedures were conducted in accordance with the ARVO statement for the use of Animals in Ophthalmic and Vision Research and after Duke University Institutional Animal Care and Use Committee protocol approval. Sprague-Dawley female rats weighing 175 to 200 g were chosen for this study. As mentioned previously, rats were considered a good animal model because they are inexpensive, easy to handle, and have a homogeneous genetic background. Furthermore, IOP measurements are easier to obtain in rats as opposed to other rodents such as mice. A total of fourteen rats were used and each rodent was sedated for the initial intracameral viral injection (day 0) with 0.1 cc of a 1:6 (Xylazine:Ketamine) mixture given intramuscularly. If necessary, repeat doses were given in 0.05 cc increments until adequate sedation was achieved. Topical proparacaine drops were instilled for additional anesthesia prior to all procedures. For IOP measurements on subsequent days, 0.1 cc of ketamine alone was given intramuscularly. After three weeks, experimental animals were sedated and anesthetized with the previously mentioned Xylazine/Ketamine mixture and then euthanized in a CO2 chamber. Eyes were then enucleated for histological analysis.
Viral delivery to the anterior segment
Viral suspension was delivered to the anterior chamber using a 31-gauge needle on a Hamilton glass syringe. All injections were monitored by direct visualization through an ophthalmic surgical microscope. The needle was inserted bevel-up through the peripheral clear cornea. A small amount of aqueous humor was expressed by pushing on the posterior lip of the wound to allow for the subsequent injection volume. Then, 2 μl to 10 μl of viral suspension was slowly inoculated while the bevel was rotated clockwise and subsequently counterclockwise to achieve an even distribution of the viral suspension in the anterior chamber. A Weck-cell sponge was used to tamponade the needle tract while the needle was removed from the anterior chamber. In this manner, a self-sealing entry site was achieved. Next, cold proparacane drops (stored at 4 °C) were placed on the cornea to induce convection currents in the anterior chamber and produce further aqueous admixture and distribution of the viral suspension. Animals were injected in one eye only and the contralateral eye used as an untreated control.
Slit lamp biomicroscopy of the anterior segment
Anterior chamber (AC) examinations were performed to scrutinize the appearance of the anterior chamber structures (corneal epithelium, endothelium, chamber depth, lens status, and AC inflammation). Sedation was achieved as previously mentioned. Examinations were performed by a board certified ophthalmologist (PC) and documented via photography on days 0, 2, 4, 6, and weekly for the following two weeks. A Topcon slit lamp biomicroscope with a camera attachment was used for this procedure.
Intraocular pressure measurements
Intraocular pressure measurements were obtained with a Tonopen XL® handheld tonometer. Pressure readings (5) were taken with the highest and lowest eliminated and the middle three recorded. Pressure measurements were taken on days 2, 6, and at weekly intervals. All pressures were compared with the contralateral, noninjected eye to account for diurnal pressure variations.
Lentiviral vectors with the elongation factor 1 alpha (EF-1α) promoter driving the expression of green fluorescent protein (GFP) was produced by Tranzyme Inc. (Research Triangle Park, NC). This is a vector based in the human immunodeficiency virus (HIV) pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) used as a reporter enhanced GFP .
Histological analysis of GFP expression
Whole enucleated eyes were fixed for 3 h in 4% paraformaldehyde in PBS, and overnight in 30% sucrose in PBS at 4 °C. GFP expression in the whole eye was visualized using fluorescence microscopy. For microscopic analysis, tissue samples from different quadrants were embedded in OCT compound (Sakura, Tokyo, Japan) and frozen over dry ice for 1 h. Sections (7 μm) of each quadrant were made using a cryostat HM505-E (Microm, Walldorf, Germany), mounted onto gelatin coated slides, counterstained with DAPI, and examined under fluorescence microscopy to analyze the distribution of GFP in the anterior chamber.
Viral particle doses and volume
To determine the optimum dose for high transduction efficiency in the TM, four different doses of lentivirus (103, 105, 107, and 109 pfu) expressing GFP were injected in the anterior chamber of eight rats. Doses equal to or higher than 107 demonstrated the best transduction efficiency. Doses lower than 105 showed low to undetectable expression.
To determine if the viral distribution in the anterior chamber was affected by the volume of viral suspension used, three rats were injected with three different volumes (2 μl, 5 μl, and 10 μl) of viral suspension at a concentration of 107 pfu. Injections using a volume of 10 μl showed the most even distribution in the anterior chamber. The smaller volumes appeared to roll on the lens and iris surfaces and accumulate in only one half of the anterior chamber angle. The larger 10 μl volume appeared to be sufficient enough to flow across the entire lens and iris surfaces and reach the majority of the angle.
Transduction expression in the anterior chamber
A lentivirus with the EF-1α promoter driving the expression of GFP was inoculated into the anterior chamber of rodent eyes. After transduction with a 10 μl volume (107 pfu), a high and an even distribution of GFP expression was observed grossly in the regions of the anterior chamber angle and corneal endothelium. Higher magnification revealed even GFP expression in the TM cells (Figure 1A,D) and the corneal endothelium (Figure 1B). Some patches of GFP expression was observed in the ciliary body and regions of the iris. Therefore, a single lentiviral injection into the anterior chamber resulted in a high expression of the reporter gene in the TM cells and corneal endothelium.
Monitoring of inflammation and corneal neovascularization
Slit lamp examination of all subject rodents revealed quiet anterior chambers without cell or flare. The corneal stroma remained clear during the course of the experiment. However, corneal neovascularization on the epithelial surface was observed in three rodents used early in this study during the dose response studies. This neovascularization appears to be mainly related to the injection procedure since modification of the injection procedure obviated its development. In affected animals, the neovascularization developed away from the puncture site over the area in which the viral particles were released into the anterior chamber. The neovascularization did not develop in later animals in which the needle bevel was rotated away from the endothelium.
The intraocular pressures of both the transduced and control eyes varied during the course of the experiment. A paired t-test was performed on the pressure differences from baseline. No significant pressure differences were observed between the transduced eyes and contralateral control eyes (see Table 1 for pressures from rats receiving 107 pfu of intracameral lentivirus). No statistically significant differences in IOP were found between transduced and control eyes. Likewise, 103 and 105 pfu did not demonstrate statistically significant different IOPs either (data not shown).
Rodents are a relatively inexpensive and easy to handle animal model whose ocular outflow pathways are similar to analogous regions of the human eye . Methods for in vivo long term genetic modification of the cells of the outflow pathway of rodent eyes can provide helpful tools to study the molecular mechanisms involved in aqueous humor outflow modulation, and those involved in the pathologic changes of the outflow pathway in glaucoma. Such methods can also be useful in testing potential therapeutic approaches for glaucoma based on genetic modifications of the cells of the outflow pathway.
Our results show the feasibility of gene transfer to the cells of the TM in live rodent eyes using lentiviral vectors. Injection of lentiviral particles in the anterior segment of live rat eyes did not result in detectable inflammation, which represents an important advantage compared to adenoviral and HSV vectors. Moreover, lentiviruses appear to be effective in transducing TM cells. However, the main technical problems we encountered in our initial experiments were the presence of variable levels of corneal neovascularization in injected eyes and an uneven distribution of the transgene expression.
We found that corneal neovascularization could be prevented by inserting the needle in a bevel-up manner through the peripheral cornea, and then rotating it 90° to ensure that the injected viral solution is not directed towards the corneal endothelium. Such orientation of the needle during the viral injections appears to be critical to prevent damage to the corneal endothelial cells that probably constitute the main factor leading to corneal neovascularization. Similarly, we found that even distribution of transduction throughout the TM could be accomplished by modifying several parameters during the viral injection. These parameters include diluting the viral suspension to a total volume of at least 10 μl and rotating the needle 180° after the injection of half of the total viral volume. Moreover, the placement of cold drops on the eye after injection may have helped distribute the viral particles by generating convection currents in the anterior chamber. All of these parameters together appear to produce a better distribution of viral particles and help increase transduction efficiency. Studies of lentiviral gene transfer to brain cells in live rodents have reported that dilution of viral particles increased the transduction efficiency by avoiding virus aggregation in concentrated viral suspensions .
The pattern of transgene expression in the anterior chamber of rat eyes transduced with lentiviruses showed important differences from that recently reported in cat eyes . While the reported injection of feline anterior segments with lentiviruses resulted in targeted expression to the cells of the outflow pathway, our results in rat eyes also demonstrated strong transduction of corneal endothelial cells, and isolated transgene expression in cells of the iris and ciliary body. The reasons for this discrepancy are not clear. A feline immune deficiency virus (FIV) driven by the cytomegalovirus (CMV) promoter to transduce TM cells was employed in that study. Loewen et al.  attributed this specific infection of the TM cells to the tropism of the VSV-G pseudotype and the convective flow of the anterior chamber. Instead of FIV, we used the HIV lentiviral vectors. However, these vectors share the same VSV-G pseudotype, which is highly effective in transducing cells of neuronal origin  like those of the corneal endothelium  and the TM .
Another difference between our study and that reported by Loewen et al.  was our use of the EF-1α promoter instead of the CMV promoter. The CMV promoter is ubiquitous and highly expressed. However, spontaneous extinction of CMV promoter activity has been reported in several cell types [33-35]. EF-1α is a housekeeping gene and is expressed at high levels in most cell types including human TM cells [36,37]. Since the EF-1α gene has indispensable housekeeping functions, expression of EF-1α is consistent over time and relatively independent from changes in cell physiology. Therefore, one possible explanation for the lack of expression in corneal endothelial cells reported by Loewen et al.  could be the preferential ability of the CMV promoter to drive expression in the TM but not in corneal endothelial cells. However, our own unpublished results using both adenoviral and lentiviral vectors containing the CMV promoter showed high expression in the corneal endothelium of injected rodent eyes, suggesting that there is not preferential expression of the CMV promoter in TM cells. Species differences in the anatomy of the anterior chamber and in the physiology of the corneal endothelial cells might also be involved in the differences of transgene expression distribution between cat and rat eyes.
In conclusion, rodents can be used as an animal model for experiments using lentiviral mediated gene transfer to the anterior chamber of the eye. Contrary to what has been observed in feline eyes, lentiviral transduction is not limited to the cells of the outflow pathway. It also extends to the cells of the corneal endothelium, iris, and ciliary body. Although our results suggest that the use of specific promoters may be necessary to target gene expression to the cells of the TM in rat eyes, we have also demonstrated that in vivo lentiviral mediated gene transfer can be a useful tool for the study of the normal and pathologic physiology of the corneal endothelium. For glaucoma studies and/or therapies, a tissue specific promoter such as the chitinase 3-like 1 promoter could be used to investigate the TM independently of the corneal endothelium .
This work was supported in part by NEI grants 5K23EY014019-02, EY05722, and EY01894. Support was also provided by the American Health Assistance Foundation (AHAF) and Research to Prevent Blindness (RPB).
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