|Molecular Vision 2004;
Received 7 November 2003 | Accepted 5 April 2004 | Published 13 April 2004
Long-term retinal transgene expression with FIV versus adenoviral vectors
Nils Loewen,1 David A. Leske,2 J. Douglas
Cameron,2 Yi Chen,2 Todd Whitwam,1 Robert D.
Simari,1 Wu-Lin Teo,1 Michael P. Fautsch,2
Eric M. Poeschla,1
Jonathan M. Holmes2
1Molecular Medicine Program and 2Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, MN
Correspondence to: Jonathan M. Holmes, Ophthalmology E7, Mayo Clinic, Rochester, MN, 55905; Phone: (507) 284-3760; FAX: (507) 284-8566; email: email@example.com
Purpose: Gene therapy for chronic retinal diseases will require long-term expression of therapeutic transgenes. Lentiviral and adenoviral (Ad) vectors are gene delivery systems with markedly different properties. Lentiviral vectors require integration into the host genome, which facilitates long-term expression, while Ad vectors remain episomal. We compared time course, location, and extent of transgene expression from replication-deficient feline immunodeficiency virus (FIV) vectors and Ad vectors in neonatal rat retina.
Methods: A dose-response study was conducted to determine the optimal subretinal dose for comparison of FIV and Ad vectors with an internal cassette expressing β-galactosidase under transcriptional control of the CMV immediate-early gene promoter/enhancer. Forty-two five-day old Sprague-Dawley rats received subretinal injections of 2 μl containing 2x103 transducing units (TU, n=14), 2x104 TU (n=14) or 2x105 TU (n=14) of FIV vector (right eye) and Ad vector (left eye). Expression was evaluated 48 h after transduction. In the subsequent long-term expression study, 60 five-day old rats received a subretinal injection of 2x105 TU FIV vector (right eye) and Ad vector (left eye). Ten pairs of eyes were analyzed at 1 week, 1 month, 3 months, 6 months, 12 months, and the remainder at 16 months. Eye cups were evaluated in a masked manner for extent of β-galactosidase expression (graded 0-5) by whole mount microscopy and by cross sectional histology.
Results: In the dose-response study, 2x105 TU resulted in consistent, widespread retinal transduction with both vectors and was selected as the dose for the subsequent study. In the long-term expression study, FIV vector resulted in a higher grade of expression than Ad at multiple single time points and produced higher overall expression when data from all eyes across the entire 16 month study were analyzed (p=0.01). Retinal expression was present at 16 months with both vectors. β-galactosidase expression was limited to the retinal pigment epithelium (RPE) until the first month, but later was also found to a lesser extent in neurosensory retina with each vector. In contrast to FIV, most Ad injected eyes showed signs of focal accumulation of macrophage-like cells with disrupted retinal architecture.
Conclusions: Both FIV and Ad vectors result in long-term transgene expression in RPE after subretinal injection. FIV vectors show more promise than Ad as delivery systems for retinal diseases since they transduce greater areas of RPE, result in less cellular infiltrate, and cause less disruption of retinal architecture. The persistent expression at 16 months of follow-up suggests that these lentiviral vectors are useful for gene therapy of chronic retinal diseases.
Ocular gene therapy offers novel approaches to treating eye diseases characterized by retinal neovascularization (e.g., diabetic retinopathy, age related macular degeneration, retinopathy of prematurity) [1-4], or chronic retinal degeneration (e.g., retinitis pigmentosa, chorioideremia, atrophia gyrata, Best's disease, Stargardt's disease) [5-12]. Several different viral vector systems have been proposed, including lentiviral and adenoviral (Ad) vectors. These vectors have markedly different biologic properties. Lentiviral vectors, such as those based on the feline immunodeficiency virus (FIV) [13-15], require integration into the host genome as an obligate part of the life cycle and have shown little direct immunogenicity or toxicity [16-18], which may facilitate long-term expression [19-21]. In contrast, adenoviral (Ad) vectors remain episomal, persist variably, undergo dilutional attrition with cell division, and are often immunogenic, all of which may result in shorter term expression [22-25].
The neonatal rat is widely used in models of retinal neovascularization, particularly for retinopathy of prematurity and diabetic retinopathy [26-32]. We have previously shown that integration is necessary for sustained expression from FIV vectors in the neonatal rat retina .
In the present study, we compared an FIV vector [18,33] pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) to an E1, E3-deleted Ad vector [34-37] to determine the extent and duration of marker transgene expression after subretinal injection. Our study involved 16 months of follow-up, which represents the longest follow-up to date in retinal transduction studies.
FIV vector production
The FIV vector used in the present study is modified from an earlier system [13,38]. Transfer vector CT26 [18,33] expresses β-galactosidase from the human cytomegalovirus (CMV) promoter [39,40] (Figure 1) and contains only the 5' 311 nucleotides (nt) of FIV gag, compared to 1249 nt in the initial transfer vector . We have previously demonstrated that only the first 311 nt of the 1352 nt FIV gag open reading frame is essential for encapsidation .
CT26 was generated by transient co-transfection of the transfer vector plasmid (pCT26), packaging construct (pCF1Δenv), and a VSV-G expression construct (pMD-G), using established transfection methods [14,15,18,20,38]. As described previously , CT26 contains from 5' to 3': the hybrid U3-substituted promoter of pCT5 , the FIV R repeat, U5 element, and leader sequence, the first 311 bp of the gag open reading frame (ORF), the Rev response element (RRE), the central polypurine tract (cPPT) , the human cytomegalovirus immediate-early gene promoter/enhancer [39,40], lacZ encoding β-galactosidase, and the 3' long-terminal repeat (LTR).
FIV vectors were produced according to a protocol  for scaled up production of lentiviral vectors by transient transfection in 10-chamber, 1 liter cell factories (model 170009 [CF10]; Nunc, Naperville, IL) with consecutive pelleting in a large volume (1.3 liter) ultraspeed rotor (model A621; Sorvall, Newtown, CT). Briefly, 293T cells were maintained at a high frequency of passage before 2.5x108 cells were seeded into one CF10 containing a total volume of 1 L DMEM (catalog number 10-017-CV; Cellgro, Herndon, VA) with 10% fetal calf serum, 100 IU/ml penicillin and 100 μg/ml streptomycin. Twenty-four hours after seeding, a transfection mix of plasmid DNA (total of 84 μg pMD-G, 252 μg CF93 and 252 μg CT26) was allowed to precipitate for 3 min at room temperature. Precipitation was stopped by adding media to a total volume of 1 L. The old media was removed from the CF10s and cells were then incubated in the transfection mix for 18 h. The transfection mix was removed and replaced with fresh media. Vector supernatants were harvested after 48 h, filtered through 0.22 μm filters and pelleted in 250 ml buckets (model 54477; Sorvall) in a fixed angle rotor (model A621, Sorvall) at 49,000x g for 2 h. Supernatants were decanted and vector pellets were resuspended in 10 ml PBS. Vector suspensions were further concentrated with a second round of ultracentrifugation at 49,000x g in 33 ml tubes in a swinging bucket rotor (Surespin, model 79367; Sorvall). Vector containing pellets were resuspended in a total of 2 ml PBS and were centrifuged for 5 min at 3,000x g to remove particulate material.
Adenovirus vector production
The adenoviral vector used in this study expressed β-galactosidase directed by the same human cytomegalovirus immediate-early gene promoter/enhancer [39,40] as the FIV vector described above. This Ad vector (Ad.CMVlacZ) [34-37] is derived from adenovirus-5 serotype and contains deletions in regions E1a spanning 1.0 to 9.2 map units and E3 spanning 78.4 to 86 map units, rendering it replication-deficient. The CMV-lacZ cassette is inserted into the E1 deletion of Ad.E1 that was parental to Ad.CMVlacZ . Ad.CMVlacZ was a kind gift of J. Wilson [34,35] to R. Simari and Z. Katusic  and was generated as described before [42,43]. Briefly, subconfluent 293 cells were infected with crude viral lysates, collecting the supernatant and purifying the viral vector by CsCl gradient ultracentrifugation repeated twice. The viral band was collected and desalted by dialysis, equilibrated with 0.01 M Tris pH 8, 0.01 M MgCl2 and 10% glycerol. Viral stocks were further purified by filtering through a 0.45 μm filter. Viral titers were determined and recorded as transducing units by infecting Crandell feline kidney (CrFK) cells. The absence of replication-competent virus was confirmed by testing the vector preparation on 3T3 cells at a multiplicity of infection of 10.
Animal experiments were conducted in adherence to the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee at our institution.
Dose responses were studied in 42 five-day old Sprague-Dawley rats (Harlan, Indianapolis, IN), which received a 2 μl subretinal injection of FIV vector into right eyes and Ad vector into left eyes. Fourteen animals received 2x103 TU of vector, 14 received 2x104 TU, and another 14 received 2x105 TU. Based on the finding of early transgene expression with FIV vector in our previous study, we assessed the extent of transduction two days after injection . Eyes of two additional non-injected animals served as grading controls.
In the long-term expression study, 60 five-day old Sprague-Dawley rats received subretinal injections of 2x105 TU FIV vector into right eyes and 2x105 TU Ad vector into left eyes. An additional 10 animals served as non-injected grading controls. At 1 week, 1 month, 3 months, and 6 months, 10 injected animals and 2 non-injected controls were sacrificed for analysis of transgene expression. Ten injected animals and 1 non-injected control were sacrificed at 12 months. Deaths by normal senescence (n=3) began to occur at 16 months, so the remaining animals (7 injected and one non-injected control) were sacrificed at that time point.
The method for subretinal injection has been described . Briefly, rat pups underwent inhalation anesthesia with isoflurane (isoflurane, USP; Abbott Laboratories, North Chicago, IL) and were placed under an operating microscope. Eyelids were gently opened with forceps. A latex membrane (standard latex exam glove) with a 1.5 mm central slit was placed over the eye and the globe was carefully prolapsed through the slit. A sclerotomy incision was made at the superior pars plana 1 mm back from the limbus with a 30 ga needle. A custom made needle (32 ga, 12° bevel, length 7 mm, Hamilton Company, Reno, NV) mounted on a 10 μl Hamilton microsyringe was directed tangentially through the sclerotomy, between the retina and sclera, and advanced to the superiotemporal subretinal space with the bevel of the needle facing the retina. A volume of 2 μl of vector preparation was slowly injected. This technique allowed confirmation of subretinal needle placement during the injection, because the injected fluid bleb with accompanying retinal detachment was visible through the sclera.
Assessment of incidence and extent of transduction
After sacrifice, eyes were enucleated and fixed for 90 min in 10% formalin at 4 °C. The cornea and lens were then removed and eye cups were stained overnight in X-gal solution to detect β-galactosidase expression. All eye cups were evaluated in a masked manner by an observer unaware of the experimental group for extent of transduction using a Zeiss stereo operating microscope (Zeiss OPMI MD; Carl Zeiss Surgical, Inc., Thornwood, NY). We used a pre-determined grading scale from 0 to 5 to quantify the extent of retinal transgene expression (Figure 2). Grade 5 was defined as confluent transduction of the entire retinal surface area, grade 4 as large confluent areas of transduction involving at least half of the retinal surface area, and grade 3 as confluent areas of transduction but involving less than half the retinal surface area. Grade 2 was defined as large areas of non-confluent transduced cells, grade 1 as the presence of isolated, discrete transduced cells and grade 0 as no detectable β-galactosidase activity.
Cross sectional histology
Two representative pairs of eye cups from each time point were embedded in paraffin, sectioned at 6 μm through the transduced area and counterstained with either neutral red or nuclear fast red. To assess the histology in a masked manner, all slides were examined by an ophthalmic pathologist (JDC) who was unaware of the experimental group.
Incidence and extent of transduction at each time point, and overall, were compared using McNemar's tests and Wilcoxon signed-rank tests.
To determine the optimum vector dose for the subsequent long-term expression study, eyes injected subretinally with 3 different doses of FIV and Ad vectors were assessed at 2 days post injection for β-galactosidase expression.
At the highest dose of 2x105 TU, all animals survived to the study endpoint. All 14 FIV vector injected eyes and 13 of 14 Ad vector injected eyes showed transduction with a median grade of 3 with each vector (p=1). In contrast, at a dose of 2x104 TU, 9 of 13 (69%) FIV vector injected eyes showed expression, compared to 5 of 13 (38%) Ad vector injected eyes. The median grade of transduction was greater in eyes injected with 2x104 TU FIV vector than those injected with 2x104 TU Ad vector, but this was not statistically significant (median grade 2 vs. 0; p=0.22). Only 2 of 12 (17%) eyes injected with 2x103 TU FIV vector and 4 of 12 (33%) eyes injected with Ad vector showed expression. One rat injected with 2x104 TU, and two rats injected with 2x103 TU, died prior to sacrifice and therefore were not analyzed. None of eyes from non-injected control animals showed transduction. Based on these results, 2x105 TU was chosen for the following long-term expression study.
Long-term expression study
Nearly all eyes (96%) through all time points of the study showed some degree of transduction (Figure 3). Analyzing data from all eyes across the entire 16 month study, FIV vector produced a higher distribution of grade of expression than Ad (Figure 3, p=0.01, Wilcoxon signed rank). Analyzing each time point separately, differences in expression grade were statistically significant at 6 months, when FIV vector transduced eyes had a higher grade than Ad eyes (p<0.05; Figure 3). At all other time points beyond one week, FIV vector transduced eyes had higher grades of transduction than Ad eyes (Figure 3), though these differences were not statistically significant due to low statistical power. For example, at sixteen months, 4 of 7 FIV vector transduced eyes were grades 3 or 4, while none of the Ad eyes achieved this degree of transduction.
Grades of expression across the 16 months suggested that FIV vector expression increased to a maximal level at 3 months, with some subsequent reduction in extent (Figure 3). In contrast, Ad vectors appeared to yield more immediate expression, peaking at 1 week, with a decline thereafter (Figure 3).
When cross sectional histology was examined, the retinal pigment epithelium showed β-galactosidase expression with both vectors at day 2 (Figure 4). In eye cups that had lower grade of transduction, the staining was greatest in the quadrant that corresponded to the site of injection. The retinae had multiple folds (not shown), similar to our findings in previous studies using subretinal injection. At day 7, expression was similar to day 2 (Figure 4).
At 1 month, extensive β-galactosidase staining could be seen throughout the retinal pigment epithelium, associated with staining of the adjacent photoreceptor outer segments (Figure 4). Some individual cells in the inner retinal layers in both FIV and Ad vector injected eyes were transduced. In Ad injected eyes, there was evidence of infiltration by large cells morphologically consistent with macrophages (Figure 4, see insert shown in 16 month section) and extracellular β-galactosidase staining was prominent. The shallow retinal folds seen earlier with both FIV and Ad persisted, presumably secondary to subretinal injection and there were isolated areas of intraretinal microcyst formation (not shown). Retinae from both groups showed areas of attenuation, consistent with resolving focal traumatic retinopathy (not shown), while the remaining retinal architecture remained intact.
At 3 months, 6 months and 12 months, FIV vector injected eyes were similar to those at 1 month with the exception of the time-dependent increase of cells in the outer nuclear layer staining positive for β-galactosidase. In Ad vector injected eyes, the intensity of RPE staining for β-galactosidase decreased over time. Transduced areas in Ad injected eyes often accumulated a focal cellular infiltrate and the previously normal retinal architecture appeared disrupted.
At the conclusion of the study (16 months), β-galactosidase positive cells were found in the RPE and occasionally in the inner and outer nuclear layers of FIV vector injected eyes. There was focal traumatic retinopathy, while the rest of the retina showed normal retinal morphology. Ad vector injected eyes still expressed β-galactosidase, but were less extensively transduced and had larger areas with disrupted retinal architecture (Figure 4).
We found similar location, extent, and duration of transgene expression in neonatal rat eyes subretinally injected with FIV and Ad vectors expressing β-galactosidase under transcriptional control of the human CMV immediate-early gene promoter/enhancer. Expression with each vector was present at 16 months post injection. There was a higher overall extent of expression with FIV, which peaked later than Ad (3 months vs. 1 week). Histology revealed greater cellular infiltrate and disruption of normal retinal architecture with Ad.
Preferential transduction of RPE over other retinal cell types by VSV-G pseudotyped lentiviral vectors has been reported [3,20,44-47]. Ad vectors appear to have a similar tropism within the retina [25,48-50]. In contrast to our present study, other laboratories have reported transduction of photoreceptors with HIV vectors . As we have noted previously , it appears that age of the animal influences tropism within the retina markedly. Animals as young as 2 days old were used when photoreceptors were transduced . In our study, occasional expression in the post-mitotic, terminally differentiated, neurosensory retina occurred at 1 month post-injection and beyond with both FIV and Ad vectors. Transduction of RPE cells is facilitated by vector uptake at their phagocytotically active, apical site . Other factors influencing differential expression between cell types include distance of and barriers to diffusion as well as varying metabolism rates of neurosensory retina and RPE.
The extent and duration of expression with FIV vectors has implications for future therapy. Lentiviral vectors may be advantageous for long-term expression over other vectors because of their ability to integrate into the host cell genome. We have previously demonstrated that a single amino acid mutation in the catalytic core domain of the FIV integrase (D66V) blocks efficient transduction, while vectors with intact integrase expressed for 7 months . Here, we have shown longer duration of transgene expression, up to 16 months. This is the longest duration yet reported for lentiviral vectors in retina, and it supports a proposed role for FIV vectors in the treatment of chronic retinal disease.
The duration of expression from a first generation Ad vector was unexpected. Marker gene expression from subretinally injected Ad vector in an immunocompetent host often lasts between 1 to 3 months [24,25,48,53]. Ad vectors lack any mechanism for integration, leaving them more vulnerable to attrition by intracellular metabolism and dilution during cell division. Although adenoviral vectors may integrate in 0.1% to 1% of infected cells [54,55], this mechanism could not explain the extent and duration observed in the present study. We speculate that the low mitotic rate of RPE cells contributes to long-term persistence of Ad vectors in this cell type.
Immunogenicity is a disadvantage of first generation Ad vectors [22,25,56-58]. We confirmed that Ad vectors induce a long-term cellular response, associated with disruption of the normal retinal architecture in our model. This retinal disruption may limit the clinical application of first generation Ad vectors in treating retinal disease. In contrast, retinal architecture was preserved in eyes injected with FIV vectors, with the exception of small areas of atrophy directly adjacent to the injection site.
Based on light microscopy, our present study suggests minimal toxicity of FIV vectors following subretinal injection. Future studies should include evaluation of function by electroretinography in sub-primate models, and electrophysiology and visual acuity in primate models.
In summary, we found persistent transgene expression up to 16 months for FIV and Ad vectors when injected subretinally in the neonatal rat. Ad induced a greater cellular response and disruption of normal retinal architecture. FIV-based vectors appear to be excellent candidates for gene transfer approaches to chronic retinal disease, particularly when lack of cytotoxicity and long-term transduction of retinal pigment epithelium is desired.
Supported by a research grant from the Knights Templar Eye Foundation (NL), NIH EY 12798 (JMH), NIH AI 47536 (EMP), a Pfizer Scholars Grant for New Faculty (EMP), Research to Prevent Blindness, Inc. (Olga Keith Wiess Special Scholar Award (JMH) and an unrestricted grant to the Mayo Clinic Department of Ophthalmology).
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