Molecular Vision 2006; 12:756-767 <http://www.molvis.org/molvis/v12/a85/> Received 22 December 2005 | Accepted 24 April 2006 | Published 11 July 2006

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Efficiency of lentiviral transduction during development in normal and rd mice

Jijing Pang,^1,2 Mei Cheng,¹ Shannon E. Haire,² Edward Barker,³ Vicente Planelles,⁴ Janet C. Blanks^1,5
 
 

¹Eye Research Institute, Oakland University, Rochester, MI; ²Department of Ophthalmology, College of Medicine, Gainesville, FL; ³Department of Microbiology and Immunology, SUNY Health Sciences Center, Syracuse, NY; ⁴Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; ⁵Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL

Correspondence to: Janet Blanks, PhD, Florida Atlantic University, Charles E. Schmidt College of Science, 777 Glades Road, PO Box 3091, Boca Raton, FL, 33431-0991; Phone: (561) 297-4310; FAX: (561) 297-2221; email: blanks@fau.edu

Abstract

Purpose: To compare the transduction efficiency of a lentiviral vector in the retina of normal mice and retinal degenerative (rd) mice following subretinal injection at various postnatal ages.

Methods: Subretinal injections of lentiviral vector (pHR-CMV-GFP, 10⁷IU/ml) were performed in normal (C57/6J) and rd mice on postnatal days P1 to P7 using a trans-scleral method and on days P14-P35 by a trans-corneal method. One to six weeks later the eyes were prepared for histological analysis. GFP positive cells were identified in retinal sections and retinal whole mounts to determine the overall extent and distribution of lentiviral transduction.

Results: Expression of GFP was observed adjacent to the injection site starting about 1 week after injection in both normal and rd mice and lasted 6 weeks (the longest period examined). In normal mice, GFP expression continued to increase and peaked around 2-3 weeks after injection with expression varying from approximately one quarter to the entire retina. GFP expression peaked earlier in rd mice injected from P1 to P7 compared to normal mice. Lenti-GFP expression decreased rapidly in rd mice older than P15. This was attributed to a period of intensive photoreceptor (PR) degeneration characteristic to this mutant. Retinal GFP expression was virtually absent in eyes injected after P14 in both normal and rd mice. Histological sections from P3 injected eyes showed GFP expression 9 days post-injection in both retinal pigment epithelium (RPE) and photoreceptor (PR) cells. GFP expression in RPE cells was stronger than that in PR cells. Both rods and cones expressed the lenti-GFP. GFP expression was limited to the RPE of normal mice if injections were performed at P14 or later. In rd mice, GFP expression in RPE was observed one week after injection at P1; GFP⁺-PR and -RPE cells were first detected 9 days after injection at P1, and 7 days after injection at P3-P7; RPE cells and occasional Muller cells around the injection site were GFP⁺ when the injection was performed at P14 or later.

Conclusions: Lentiviral-mediated GFP transduction of RPE was efficient and sustained at all ages examined in both the normal and rd mouse. Trans-scleral, subretinal injection of lenti-GFP during the first postnatal week produced age-dependent transduction of PR cells in both mouse strains. Lenti-GFP expression was absent in both mouse strains if injections occurred after P14. There was a dramatic decrease in the transduction efficiency in rd mouse retinas corresponding to the degeneration of PR cells. However, the early stages of retinal degeneration in rd mice appeared to increase the transduction efficiency of PR cells. These data suggest that both age and degree of PR degeneration are important parameters to consider when designing gene therapy experiments or protocols.

Introduction

Retinitis pigmentosa (RP) is a genetically and clinically heterogeneous group of retinal degenerative diseases, considered to be the leading cause of inherited blindness affecting approximately 1 in 3,500 people [1]. Clinical manifestations of RP include impaired light adaptation, night blindness, progressive loss of visual field, bone spicule pigmentation in peripheral retinas and abnormal electroretinogram [2]. Visual impairments in RP are caused by the progressive degeneration of retinal photoreceptor (PR) cells, which is triggered by a mutation of certain genes. To date, over 90 genes have been identified that cause RP. The majority cause photoreceptor defects when mutated [3], but mutations causing dysfunction of retinal pigment epithelial cells (RPE) are also prevalent [4]. Many of the genes expressed in photoreceptor cells encode enzymes of the phototransduction cascade, photoreceptor-specific structural proteins, or transcription factors [5].

Naturally occurring mutations of the β subunit of phosphodiesterase (β-PDE), an enzyme that degrades cyclic guanosine monophosphate (cGMP), in rod PR cells have been identified in human autosomal RP [6,7] and in mutant rd mice [8,9]. Rd mice are one of the best studied animal models of RP and are characterized by a rapid loss of rod photoreceptor cells between postnatal day (P) 8 and P15, followed by a more gradual degeneration of cones [10]. The peak of PR cell apoptosis is observed by P13, and by P16 almost the entire population of PR cells has been lost [11]. It has been shown that transfer of a wild type normal gene can correct retinal degeneration in the rd mouse [12].

Delivery of a corrective gene to the rd mouse eye has been partially successful using adenoviral (Ad), adeno-associated virus (AAV) and a lentiviral vector. Ad was used to deliver βPDE to rd mice and delayed cell death for 6 weeks [13]. A similar approach was tried using AAV with evidence of a therapeutic effect [14]. Improved levels of histological rescue were reported using a gutted adenovirus which slowed the degeneration for 12 weeks in the rd [15]. The longest documented rescue in the rd retina was obtained using a lentivirus carrying βPDE [16]. In the latter study, rhodopsin expression indicated that about 20% of the PR cells were rescued although photoreceptor outer segments were not convincingly demonstrated in the published figures.

The replication-defective lentiviral vectors based on the human immunodeficiency virus type 1 (HIV-1) offer the advantage that they do not encode viral proteins that may elicit an immune response. Moreover, unlike other retroviruses which transduce dividing cells exclusively, the lentiviral vector can integrate into the chromosomes of both dividing and nondividing cells. Lentiviral vectors drive stable, long-term transgene expression in both the retina and the brain [17,18], transfect adult, mature neurons and possess a larger cloning capacity (approximate 9 kb) than AAV (6 kb). Because of its many advantages, lentiviral vectors are actively being pursued as potential vectors to rescue degenerating PR cells. The effect of the injection site on cell transduction has been studied [19,20] and the tropism of different pseudotyped lentiviruses has been documented [21]. Despite the existing literature, there is still controversy as to the extent of PR transduction with lentiviral vectors. This study was designed to determine if mature PR cells could be transfected and to determine the optimal postnatal age for lentiviral-mediated gene therapy of mouse models with PR and/or RPE degeneration.

Additionally, it has been assumed that virus-mediated gene expression has higher transduction efficiency in PR degenerative retinas [22-24]. Recently we verified in an in vitro, retinal explant study that retinal degeneration in rd mice can dramatically increase adenovirus-mediated transduction efficiency [25]. This paper focuses on the effect of in vivo retinal development on lentivirus-mediated transduction efficiency in rd mice.

Methods

Animals

Normal (C57BL/6J) and retinal degenerative (rd) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and bred in our institutional animal facility. All animals were treated, maintained and sacrificed in accordance with the ARVO statement for the use of Animals in Ophthalmic and Vision Research and with Federal, State and local regulations. Ten to twenty mice received subretinal injections of the vector on each of the following days: P1, P3, P5, P7, P14, and P35. An approximate total of 90 normal and 100 rd mice were used in this study.

Plasmid constructs

The transfer vector, pHR'-CMV-GFP (lenti-GFP) was described previously [26]. This vector was constructed by replacing the lacZ reporter gene in pHR'-CMV-lacZ [18] via digestion of the latter with BamHI and XhoI, followed by fill-in of XhoI, with a BamHI to HpaI fragment of pEGFP-N1 (Clontech, Palo Alto, CA) [27].

Viral vector production and titration

The transfer vector, pHR'-CMV-GFP, (D102-mHSA, D102-vprH or D102-vprA) was cotransfected with vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors [28] and pCMV-DR8.2Dvpr [29] using the calcium phosphate-mediated transfection. Virus was collected at 48, 72, 96 h post-transfection. The harvested supernatants were pre-cleared by low-speed centrifugation at 2,000 rpm and pelleted by ultra-centrifugation at 25,000 rpm in a Discovery 100S centrifuge with a Surespin 630 rotor (Sorvall, Newton, CT). Virus pellets were re-suspended in fresh tissue culture medium and frozen at -80 °C. Vector titers were approximately 1.5x10⁷/ml, which were measured by the infection of HeLa cells as described below, followed by flow cytometric analysis of cells positive for the reporter molecule, GFP. Vector titers were calculated as (FxC₀/V)xD where F represents frequency of GFP⁺ cells by flow cytometry, C₀ represents total number of target cells at the time of infection, V represents volume of inoculum, and D represents virus dilution factor. Virus dilution factor used for titrations was ten (e.g., D=10). Total number of target cells at the time of infection was about 10⁶. Pseudotyped lentiviral vectors produced by the above methods have been shown to transduce a number of cell types in vitro and in vivo [18,30].

Subretinal injection

Neonatal mice aged P1 through P3 were anesthetized by chilling on ice for 3 to 5 min until they became immobilized. The pups were placed on a pad of paper towels with an ice bag beneath to maintain anesthesia. P5 and older mice were anesthetized by an intraperitoneal injection of ketamine (72 mg/kg)/xylazine (4 mg/kg). For the mice older than P14, 1% atropine eye drops (to dilate the pupil for a longer time and to reduce post-surgical inflammation) were given three times at hourly intervals on injection day. This was followed by administering repeated topical 2.5% phenylephrine hydrochloride eye drops just before and after the injection of anesthetics. Subretinal injections were performed on C57BL/6 mice at P1 to P7 with 0.5 μl of pHR-CMV-GFP injected into one eye using published methods [13,14] with minor modifications. The eyelid was opened by blunt separation with forceps and a slight amount of periocular pressure applied to slightly proptose the eyeball. The eyelids served to hold the eyeball out of orbit. The conjunctiva was cut using the sharp edge of a 30-gauge needle to expose the underlying sclera. The conjunctiva adjacent to the cornea was grasped and rotated with forceps to allow optimal exposure of the injection site. A hole was made in the sclera with the tip of a 30 1/2-gauge disposable needle. The tip of a 33-gauge blunt needle, mounted on a 10 μl Hamilton Micro Syringe, was inserted into the potential subretinal space. An assistant slowly released the vector into the eye by placing slight pressure on the plunger of the syringe until 0.5 μl of vector was delivered. Subretinal injections were made under direct observation aided by a dissecting microscope at 6x magnification. For subretinal injections on mice at P14 to P35, a hole within the dilated pupil area was made through the superior cornea with a 30 1/2-gauge disposable needle. A 32-gauge unbeveled blunt needle mounted on a 10 μl Hamilton syringe was then introduced through the corneal opening, avoiding the lens and penetrating the retina. One μl of vector suspension with 1% fluorescein was then slowly injected subretinally. The retinal area injected was visualized by fluorescein-positive subretinal blebs illustrating a slight retinal detachment. Immediately following injection of the vector, one drop of 1% Atropine and a small amount of Neomycin & Polymyxin B Sulfates & Dexamethason Ophthalmic Ointment were applied to the eye. This post-operative care of drops and ointment was administered once/day for the first 3 days. Animals were sacrificed 1-6 weeks later, the eyes enucleated, the cornea and lens removed and the eyecup fixed with 4% paraformaldehyde in phosphate buffer.

Analysis of lenti-infectivity in whole mounts and tissue sections

To document the extent of lenti-GFP distribution across the retina, retinal whole mounts were prepared from some eyecups by removing the sclera and choroid, together with the retinal pigment epithelium (RPE). The retina was cut at 3, 6, 9, and 12 o'clock to flatten the whole mount for photography using a fluorescent microscope. After the whole mounts were photographed, they were embedded in acrylamide [31] and sectioned with a cryostat. Other eyecups were fixed and embedded in acrylamide and sectioned with a cryostat for microscopy. All cryosections were analyzed using a fluorescent microscopy to identify GFP⁺ retinal cells.

Images were collected on an Olympus AX 70 photoscope equipped with a magnifying digital camera. Photographic plates were assembled using Adobe Photoshop with minor adjustments for contrast, brightness and color tone.

In order to evaluate the variability of transduction at different ages of normal and rd mice, retinal whole mounts were photographed and the FITC-positive area was classified into 5 different levels depending on the intensity and pattern of fluorescence (see Table 1).

Results

All eyes injected with lenti-GFP vector were free of clinical anterior or posterior segment inflammation. The retinal bleb created by trans-corneal subretinal injection in older mice subsided gradually with the retinal reattaching usually completely within a day or so. No retinal bleb could be visualized following trans-scleral, subretinal injection in neonates due to the opaque lens following anesthesia by ice. Furthermore, corneal opacity, cataract formation and retinal damage were often observed, perhaps due to immature eyes and the technical difficulty in injecting such small eyes.

GFP expression in retinal whole mounts following subretinal injection

Normal mice: GFP expression was variable following subretinal injection at all ages from P1 to P7. Little GFP expression was evident in retinas from mice injected on postnatal day 1 (Figure 1A). Expression was first detected around the injection site 9 days after injection at P3 (P3 [date of injection] +9 [number of days following injection], Figure 2A). GFP expression was at its peak between 2-3 weeks after injection at P3 and was present throughout most of the retina (P3+19, Figure 2B); GFP expression was maintained for 6 weeks (the longest period examined; P3+42, Figure 2C). At P5, GFP expression appeared about 1 week following injection (data not shown), and was still present almost 3 weeks after injection (P5+19, Figure 2D). At P7, GFP expression also appeared about 1 week following injection (P7+7, Figure 2E) but only around the injection site. GFP expression also peaked 2 weeks following injection at P7 (P7+14, Figure 2F). GFP expression was not observed in retinal whole mounts when injections were performed at P14 or older in normal mice (Figure 2G, P14+14). In most cases, GFP expression occupied about 1/4 of the retina but sometimes expression was observed over a larger area (Figure 2B,D,F). "Clumps" of GFP⁺ cells were often observed in the retinal whole mounts following lentiviral injection in normal mice (arrows, Figure 2C,F).

Rd mice: The pattern and extent of GFP expression in rd mice differed from normal mice both temporarily and spatially. No GFP expression was observed in the first week following injection at P1 (Figure 1B) but was evident by 2 weeks (P1+14, Figure 3A). Scattered weak GFP expression usually started around the injection site 7 days after injection at P3 (P3+7, Figure 3B), and was more intense and widespread, covering about one-quarter of the retina after 9 days (P3+9, Figure 3C). GFP expression appeared around the injection site one week after injection at P5 (P5+7, Figure 3D), increased by 8-10 days post-injection (Figure 1B) and appeared in limited retinal areas at 2 weeks (P5+14, Figure 3E). The precipitous decrease in GFP expression in animals sacrificed after P16 may be due to the massive PR degeneration between P12-P16 in therd mouse. GFP expression was evident a week after injection at P7 and occupied approximately one quarter of the retina (P7+7, Figure 3F). There was no apparent GFP expression in retinal whole mounts from rd animals injected at P14 or older (P14+21, Figure 3G). Please note that GFP expression in P3+9 rd retinas (Figure 3C) was more intense than in P3+9 retinas of normal mice (Figure 2A). GFP expression was present in reasonably large areas of the retina and did not appear in "clumps" as seen in normal retinas following injection of the lentivirus vector.

An analysis was performed of the relationship between the transduction efficiency at specific days after injection on various postnatal days in both normal and rd mice (Figure 1). There was some variation in transduction efficiency from animal to animal, however, a common pattern of developmentally regulated transduction efficiency was observed. In normal mice, GFP expression was evident by 1 week after injection in P5 and P7 animals but was not detectable until 9 days when the injection was performed in P3 animals. Expression peaked between 2 and 3 weeks post-injection. No transduction of lentivirus was evident in P14 or P35 animals.

Transduction efficiency in the rd mouse differed substantially from the normal since the peak of transduction was achieved earlier in the rd (i.e., between 1-2 weeks, compare Figure 1A,B). Expression of GFP did not last long in the rd due to the massive degeneration of the PR cells that occurs between P12-P16.

GFP expression in sections prepared from retinal whole mounts and eyecups

Normal mice: Histological examination of normal mice showed no GFP fluorescence either in RPE or PR cells for the first 2 weeks following injection at P1 (data not shown). Scattered weak GFP⁺ RPE cells were observed 3 weeks following injection at P1 (data not shown). In mice injected at P3, weak GFP expression was detected only in RPE cells 7 days after injection (data not shown); by 9 days following injection at P3, GFP expression was evident in both RPE and PR cells (Figure 4A, P3+9) with greater GFP expression in RPE cells than in PR cells; by 19 days after injection at P3, PR cells displayed the most GFP expression (Figure 4B-D, P3+19) in sections prepared from a retinal whole mount (Figure 2B). GFP expression was mainly located in the outer nuclear layer and inner and outer segments of PR cells (Figure 4B). Both rods and cones appeared to be transfected with the lenti-GFP (note the cone at second arrow from the left side of the picture in Figure 4B). The cone in this figure can be identified by the position of its nucleus (directly below the outer limiting membrane) and by the size of its synaptic terminal (it has a larger diameter pedicle compared to the spherule of the rod terminal next to it). In normal mice, GFP⁺ cells remained for at least 6 weeks following subretinal injection (Figure 4E, P3+42; Figure 4F, P5+43).

Increased stability and higher efficiency of vector delivery was obtained using the trans-corneal approach. For subretinal injections in P14 or older mice GFP, expression usually appeared 1 week following injection (data not show) and lasted for at least 6 weeks (Figure 4F,G, P35+42). GFP expression was maintained throughout the whole retina 6 weeks following injection at P35 (Figure 4G). Primarily RPE cells were transfected in normal mice injected at the older ages (Figure 4H, P35+42).

Rd mice: Weak GFP expression appeared only in scattered RPE cells 7 days following injection at P1 (arrows, P1+7, Figure 5A); and by 9 days, both GFP⁺ RPE and PR cells were observed (P1+9, Figure 5B), with more intense RPE expression. Injections at P3 produced more GFP⁺ RPE and PR cells at 1 week (P3+7, Figure 5C). Although GFP expression was present in P5+7 mice, less and weaker GFP expression was observed in PR cells than in RPE (P5+7, Figure 5D) cells. At this stage of degeneration, there were many PR nuclei, which were GFP⁺. Eyes injected at P7 with enucleation a week later expressed GFP primarily in the RPE (few PR cells were present in the degenerating outer nuclear layer [P7+8, Figure 5E]). Besides GFP⁺ RPE cells throughout the eyecup (Figure 5F), GFP⁺ Muller cells were also observed 6 weeks following injection at P35 (Figure 5G), but only around the injection site. GFP expression in Muller cells was not observed in normal mice following lenti-GFP injection. GFP expression was observed in RPE cells from 1 to 6 weeks following injection at various ages in both rd and normal mice.

Light microscopy at P12 showed a dramatic decrease in the number of PR cells in the rd retina (Figure 6B) compared to the aged-matched normal retina (Figure 6A), which is consistent with results published in the literature [10,11].

Discussion

Several studies have shown that gene therapy is a promising technique to correct gene defects affecting the retina or to rescue retinal cells, including PR or RPE cells [16,32-41]. In early studies, a recombinant adenovirus was employed for in vivo retinal gene transfer, and showed a transient rescue of PR cells in rd mice [42-44]. However, adenoviral vectors can evoke a host immune response resulting in a transient gene expression. As a result other alternative vectors are under investigation for future retinal gene therapy. These vectors include recombinant adeno-associated virus (rAAV) [45-47] and human immunodeficient virus (HIV)-based lentiviral vectors [32]. Partial rescue of PR cells from degeneration occurred in about 40% of rd mice in experiments using a lentiviral vector [16]. Both lentiviral and adeno-associated viral (AAV) vectors may be suitable for effective treatment of retinal diseases since they both reportedly transduce dividing and nondividing neurons and are able to sustain transgene expression [18,32]. Although they can infect both developing and mature PR cells, AAV vectors have a limited carrying capacity. Therefore, lentiviral vectors are advantageous when the size of the transfer gene size is larger than 4.4 kb.

Lentiviral vectors were first used in retinas to transfer the GFP reporter gene into the retina of rd mice under the control of the CMV promoter. In this study the pHR' vector described by Miyoshi et al. [32], was able to express the transgene very early and to sustain expression of the transgene for over 6 months. However, only P2-P3 mice were used in the previous study raising the question whether different age groups might exhibit varying propensities for transgene expression. Indeed, in the present study, we confirmed that transduction varies with the developmental state of the retinas as evidenced by lentiviral vector transduction of developing PR cells when the subretinal injection was given to neonatal pups. The lentiviral vector driven by a CMV promoter and encoding the GFP reporter gene could be used to transduce developing PR cells in normal mice following subretinal injection from P1-P7. Earlier GFP expression was observed after injection at P5 or P7 compared to injections performed at P1 or P3 (Figure 1A). Although weakened, GFP expression was maintained in PR cells for up to 6 weeks (the longest period examined) following injection. However, it was rare to find PR cells transduced when subretinal injections were performed in P14 or older animals. Even though PR cells were undergoing degeneration at this time, no lentiviral vector was taken up in P14 rd animals. Although AAV with a chick β actin (CBA) promoter or a cone-specific promoter can transduce cones very well (unpublished data), it has been reported that transduction of cones failed to be detected even in areas where nearly 100% of the rods were transduced using rAAV with CMV driving GFP expression [48]. In contrast, we provide evidence that both cone and rod photoreceptor cells can be transduced with the lenti-CMV vector used in this study.

GFP expression in RPE cells was not observed in retinal whole mounts prepared from either older normal or rd mice since the transduced RPE cells "float off" when preparing retinal whole mounts. It is difficult to keep the RPE cells on the surface of the neural retinal when separating the retina from the eyecup. Therefore, the data shown in Figure 1 indicate the transduction efficiency in PR cells, but not RPE cells. RPE cells were transduced at all ages studied with stable expression for up to 6 weeks. This observation confirms reports in the literature that the tropism RPE cells is usually much higher than that of PR cells following subretinal injection of a variety of viral vectors, including those based on adenovirus [22,24], lentivirus [49] and AAV [50]. It should be noted that intravitreal injections of a lenti-GFP vector driven by a CMV promoter also targeted RPE cells without transducing retinal neurons [51].

There was only weak and scattered GFP expression in RPE cells following injection at P1. This may be related to the technical difficulty of injecting neonatal eyes and/or the state of development of the P1 retina. Surprisingly, the highest level of GFP expression was observed in sections and retinal whole normal P3+19 mice. This may be related to a very successful injection and/or the developmental status of the retina at this age.

Our data, together with results presented in the literature, raise doubts regarding the efficiency of lentivirus-mediated gene expression in nondividing, mature PR cells. In fact, it is possible that some of the expression seen in mice injected during the first postnatal week may be occurring in PR cells undergoing cell division. Using thymidine labeling, several laboratories have shown that rod PR cells are still dividing in the peripheral retina until the end of the second postnatal week [52-54]. It is possible that more efficient transduction could have been accomplished if we had used a cell-specific opsin promoter, as in one of the original lentiviral studies in retinas [32] rather than the universal CMV promoter used in other gene therapy studies. Future studies should focus on the efficiency, tissue specificity and longevity of opsin-promoter-driven lentiviral vectors.

Our results provide additional information regarding the efficiency of gene transfer during retinal development and degeneration. Our earlier, in vitro study using adenovirus-mediated transduction showed increased efficiency in retinal explants from rd mice compared to normal mice [25]. Adenovirus-mediated reporter gene expression in retinal whole mounts increased dramatically following major retinal degeneration after P14 in rd mice, while there was little expression in age-matched normal C57 BL/6J mice [25]. In contrast, lentivirus-mediated gene expression observed in this study showed little increase following massive retinal degeneration in rd mice compared to aged-matched normal mice. Adenoviral-mediated gene transfer occurs via specific receptors on the cell surface before any "down stream" event happens. It is possible that the specific receptors for adenovirus and lentivirus are located in different layers of the retina, which may lead to different transduction efficiencies.

The earliest signs of GFP expression were detected adjacent to the injection site in both normal and rd mice. Increased transduction at the injection site may be due to increased viral uptake because of the weakened physical barrier following subretinal injections. We did note that GFP expression peaked earlier in rd (Figure 3C, P3+9) compared to normal mice (Figure 2B, P3+19) and was more intense under the similar conditions when advanced PR degeneration had not occurred in the rd retina (Figure 3C versus Figure 2A and Figure 3F versus Figure 2E). Besides GFP expression in RPE cells, GFP⁺ Muller cells were observed around the injection site, but only in rd mice. This data suggest enhanced transduction efficiency in degenerating retinas, which may be related to the weakened physical barrier due to PR loss and injection and/or the Muller cell hypertrophy noted in rd mice. Hypertrophied Muller cells in the rd mouse express glial fibrillary acidic protein which correlates to the increase in PR cell death in this mutant [55].

The trans-scleral, subretinal injection method appears to be a practical method to deliver the vector to PR cells in neonatal mice, although it is not perfect. In most cases, only about one quarter of the retinas in neonatal mice aged P1-P7 were transduced using this method. Because of the size of the mouse eye at this age, several complications such as corneal opacity, cataract and retinal damage can occur with the trans-scleral injection method. This relatively small portion of the transduced retinas together with the possible damage due to the subretinal injection might be the reason why previous studies of the rd mouse showed so few PR cells were rescued [13,16] and only an in vitro ERG was detected [14].

Subretinal injections were more successful using a trans-corneal approach in mice P14 (with the eyes open) or older. It is impractical to perform a trans-corneal, subretinal injection in neonates. Few complications were observed in both normal and rd mice with one subretinal injection at P14 or older. The entire RPE could be transfected. Long term, widespread and stable expression in PR cells was accomplished following trans-corneal subretinal injection of an AAV vector in adult mice [14,45,56,57]. Our results, together with the literature [19] suggest that lentiviral vectors are good candidates for gene therapy targeting RPE cells and/or PR cells if the PR degeneration is slower, such as in the rd mutant [33,58,59] or when the genetic lesion is in the RPE such as in the rd 12 mutant mouse [60] or RCS rat [61]. Considering the technical difficulties involved in the trans-scleral, subretinal injection in neonatal mice, lentivirus-mediated gene therapy targeting PR cells that have slow degeneration will have to deal with the problem of mature PR cell transduction. A possible alternative is the use of AAV vectors. However, as our understanding of gene targeting via cell specific promoters is enhanced, the utility of lentiviral vectors may increase. Other experimental paradigms may be tried to increase PR cell transduction. For example, recently it was shown that simultaneous injection of neuraminidase X and a lentiviral vector increased the number of transduced PR cells significantly (about five-fold) [62].

In conclusion, our results emphasize the importance of considering the age of the animal and the degree of photoreceptor degeneration when designing gene therapy protocols.

Acknowledgements

This study was supported by NIH grants (EY12164, EY05230 and AI049075) and grants from the Knights Templar Foundation and the Midwest Eye Banks and Transplantation Center (MEBTC). The authors wish to thank Drs. Kathy Dorey, and Mingwu Wang for carefully reading the manuscript and Dr. Christopher Dougherty for assistance with publication figures. Part of this research was presented in abstract form at the annual meeting of The Association for Research in Vision and Ophthalmology, May, 2002, Ft. Lauderdale, FL.

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