An ex vivo gene therapy approach in X-linked retinoschisis

Disease model

Homozygous female Rs1h knockout (KO) and hemizygous male Rs1h KO animals were obtained from the laboratories of Dr. R. Molday (University of British Columbia). Offspring were genotyped with PCR to verify the genetic status using two sets of primers. PCR conditions: Denaturation - 30 s, 94 ºC. Annealing - 30 s at 60 ºC; Extension - 45 s at 72 ºC. Final extension cycle extended to 10 min at 72 ºC. Cycles repeated 35 times.One set (forward: 5′-TGA GGA CCC CTG GTA CCA GAA-3′; reverse: 5′-CCA TCT CAG GCA AGC CAG G-3′) was designed to amplify a 260 bp region of the wild-type Rsh1 gene. The same forward primer was used in combination with a different reverse primer targeting LacZ (5′-CAA GGC GAT TAA GTT GGG TAA C-3′) to detect the mutant Rs1h gene (product size 180 bp) [22]. Homozygous female and hemizygous male offspring were used for this study. The animals were housed under standard conditions (25 °C; 10% relative humidity, and a 12-h:12 h light-dark cycle) and had free access to food and water throughout the experiment. These studies were approved by the University of British Columbia Animal Care Committee in Canada and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Cloning of RS1 cDNA

For the constitutive expression cassette, the human RS1 cDNA (gift from Dr. R. Molday) was cloned into the pIRES2-DsRed2 vector (Clontech, Mountain View, CA) driven by a constitutive cytomegalovirus (CMV) promoter. This plasmid also carries the neomycin-resistance gene for the selection of transfected cells with the antibiotic Geneticin (G418). This plasmid was used to transform competent DH5α E. coli cells (New England Biolabs, Whitby, Canada). The transformed bacterial cells were cultured overnight at 37 °C in Luria Broth media (Thermo Fisher Scientific, Ottawa, Canada) containing 50 µg/ml kanamycin (Thermo Fisher Scientific). Plasmid isolation was performed using the PureYield^TM Plasmid Maxiprep System (Promega, Madison, WI) according to the manufacturer’s instructions. The presence of the RS1 cDNA in the plasmid was confirmed by restriction digestion and subsequent 1% agarose gel electrophoresis.

For the inducible expression cassette, a Tet-On system (Clontech) was used that contained two plasmids: pTRE3G-BI-mCherry and pCMV-Tet3G. The human RS1 cDNA was cloned into the pTRE3G-BI-mCherry vector. This plasmid system carries the hygromycin-resistance gene for the selection of transfected cells with the antibiotic Hygromycin (Hyg). The other plasmid (pCMV-Tet3G) carries the neomycin-resistance gene for the selection of transfected cells with the antibiotic Geneticin (G418). Both plasmids were amplified using DH5α E. coli cells following the above-described procedure. The presence of RS1 cDNA in the pTRE3G-BI-mCherry plasmid was confirmed by restriction digestion and gel electrophoresis.

Cell culture

MSCs were isolated from inguinal adipose tissue (AMSCs) as previously described [14]. Briefly, the tissue from P21 C57BL/6J mice was macerated with surgical scissors in 3 ml of 1 mg/ml Collagenase I (Sigma-Aldrich, Oakville, Canada) in Dulbecco’s PBS (Life Technologies, Burlington, Canada) and incubated at 37 °C for 1 h. Collagenase-dissociated tissue was filtered through a sterile 40 µm cell strainer (Stemcell Technologies, Vancouver, Canada) into a 50 ml sterile tube and centrifuged at 1,000 ×g for 10 min. The supernatant was removed, and the cell pellet was resuspended in MesenCult MSC Basal Medium (Stemcell Technologies) supplemented with 15% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies). The cells were plated in a six-well dish and allowed to adhere for 48 h in a 37 °C incubator with 5% CO₂. Upon reaching confluency, the cells were passaged to a T-75 cell culture flask as previously described [14].

WeriRB human retinoblastoma (Weri-Rb-1) cells were obtained from American Type Culture Collection (Manassas, VA). These cells were used after two passages as a control to show the size of the human RS1 protein on western blot as previously authenticated [23]. The cells were thawed and maintained in suspension culture in RPMI 1640 media (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). The cells were plated in a T-75 cell culture flask and incubated in a 37 °C incubator with 5% CO₂.

MSC transfection with RS1 cDNA containing expression vector

Mouse MSCs were electroporated using the Gene Pulser Xcell system (Bio-Rad, Mississauga, Canada). Briefly, MSCs from an early passage (3–5) were dissociated with 0.05% Trypsin-EDTA (Life Technologies), counted, and collected by centrifugation. One million cells were resuspended in 500 µl of Gene Pulser electroporation buffer (Bio-Rad) and transferred into a 0.4 cm electroporation cuvette (Bio-Rad). For the constitutive expression, 100 μl pIRES2-DsRed2-RS1 plasmid (1 μg/μl) was mixed thoroughly with the MSCs containing electroporation buffer. A square wave pulse of 220 V for 25 msec was applied to the cells, which were then quickly plated in a six-well dish with 2 ml MesenCult MSC Basal Medium and incubated at 37 °C. The medium was replaced 24 h later with MSC media containing 200 µg/ml G418 (Geneticin, Life Technologies) for selection over a 2-week period. Transfection was confirmed by visualization of the reporter gene DsRed2, using a Zeiss LSM 510 META confocal laser scanning system (Zeiss Canada, Toronto, Canada).

Expression and secretion of RS1 from transfected cells were assessed with an enzyme-linked immunosorbent assay (ELISA) as described previously [14]. Briefly, 1,000 transfected cells in 200 µl of culture media were plated into a 24-well tissue culture dish. Five days later, the media were collected from each well, and the cells were lysed with 200 µl of lysis buffer (10 mM Tris-HCl pH 7.4, 2 mM EDTA, 150 mM NaCl, 0.875% Brij-96, 0.125% NP-40, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 174 µg/ml phenylmethane sulfonyl fluoride [PMSF]). Lysates were collected in 0.5 ml tubes and were subjected to three freeze–thaw cycles. After brief centrifugation for 1 min at 10,000 ×g, the supernatant was collected. Using 100 µl of cell lysate or media, RS1 expression and secretion were measured using ELISA kit (USCN Life Sciences Inc., Wuhan, China) following the manufacturer’s instructions. Visualization of the chromogenic substrates was measured at 450 nm using a POLARstar Omega plate reader (BMG LABTECH, GmbH, Ortenberg, Germany). All samples were analyzed in duplicate (n = 3), and the results were calculated as ng/million cells/day.

For the inducible system, two rounds of transfection were performed. First, 100 μl of pCMV-Tet3G (1 μg/μl) plasmid was used to transfect the mouse MSCs. Successfully transfected cells (TetMSCs were selected using G418 (200 µg/ml) for 2 weeks followed by rapid expansion. A second round of transfection was performed using the pTRE3G-BI-mCherry vector with RS1 cDNA (1 μg/μl) on the TetMSCs following a similar electroporation process. Transiently transfected cells were observed by imaging the reporter gene mCherry using confocal imaging. Hygromycin (Thermo Fisher Scientific) selection media (200 µg/ml) were used to select the transfected cells (inducible MSCs) for 2 weeks, and then RS1 secretion was measured as described above.

Intravitreal injection

For the single-injection protocol, expanded AMSCs, constitutive MSCs, and inducible MSCs were resuspended in Dulbecco’s PBS (ThermoFisher Scientific, Product No: 14190250, Burlingham, Canada) to give a final concentration of 50,000 cells/μl. A 30 gauge needle was used with the Hamilton was used to inject 2 μl of the cell suspension (1× 10⁵ cells) into the vitreous cavity of the left eye of the P21 Rs1h mice. The injection site was about 1 mm behind the limbus. The animals were anesthetized by inhalation using 3% isoflurane. Five groups of animals received constitutive RS1-secreting MSCs, inducible RS1-secreting MSCs, unmodified AMSCs, or culture media (vehicle) or were untreated. Immunosuppression was not used in any animals. Data were collected at 2-, 4-, and 8-week post-injection time points. For the multiple injection protocol, the first injection was performed at P30, followed by injections at P60 and P90. There were three cohorts in this study: inducible MSCs, constitutive MSCs, and untreated. Data were collected at P120.

Topical doxycycline administration

To test the penetration of doxycycline into the retina, Rs1h mice (n = 3) were treated with topical Doxycycline Hyclate (doxycycline; Sigma-Aldrich) for 7 consecutive days. Two concentrations of doxycycline were prepared (5 and 10 mg/ml) using PBS (Life Technologies). Doxycycline (50 µl) was applied to the left eye of each animal using a dropper. For the in vivo studies (intravitreal inducible MSCs), 50 µl of 5 mg/ml doxycycline was topically administered to all animals 1 day after the injection and continued once daily until the sampling date.

Electroretinography

Mice were dark-adapted overnight, then anesthetized with an intraperitoneal injection of xylazine (16 mg/kg) and ketamine (120 mg/kg), and maintained on a heating pad. Using topical 0.5% proparacaine hydrochloride (Bausch and Lomb, Rochester, NY), the cornea was anesthetized, and the pupils were dilated with 2.5% phenylephrine and 1% atropine. A drop of 2% hydroxy-propyl-methyl-cellulose was placed on each cornea to keep it moist. Electroretinograms (ERGs) were recorded using an Espion V5 Electroretinography Console plus ColorDome Mini-Ganzfeld stimulator (Diagnosys LLC, Lowell, MA) with corneal contact lens electrodes. Scotopic ERGs (reflecting rod function) were recorded using white flashes of intensity 2.25 cd.s/m². Photopic ERGs (mixed rod and cone function) were recorded with the background illumination set at 30 cd/m² and white flashes of 3.0 cd.s/m². The average scotopic b-/a-wave ratio was calculated. The sample size was six for all cohorts for the single- and multiple-injection studies.

Optokinetic tracking

To examine visually driven behavioral responses, optokinetic tracking (OKT) was used to measure the spatial frequency thresholds under photopic (light-adapted) conditions [24]. A vertical sine wave grating (100% contrast) was projected as a virtual cylinder in three-dimensional space on computer monitors arranged in a quadrangle around a testing arena (OptoMotry; CerebralMechanics, Lethbridge, Canada). Unrestrained mice were placed on the elevated platform at the center of the equipment. An observer used a video monitor to ensure that the virtual cylinder was kept centered on the animal’s head and recorded head movements in response to cylinder rotation. The highest spatial frequency that resulted in a head movement response was documented. The sample size was six for all cohorts for the single- and multiple-injection studies.

Histology

Animals were euthanized with CO₂ asphyxiation. The eyes were enucleated and marked at the 12 o’clock position, by leaving a piece of white conjunctiva muscle for orientation. The eyes were immersed in 50% Karnovsky fixative for 1 h, and the anterior segment was removed by excision at the ora serrata. The posterior eyecups were embedded in paraffin, cut sagittally to a thickness of 5 μm, and counterstained with hematoxylin and eosin. Schisis cavities within the inner nuclear layer were analyzed. To standardize the schisis area measurement of different eyes, only sagittal sections that included the optic nerve were assessed. Images were overlaid with a 200-μm-long rectangular template using Adobe Photoshop CS2. This template was positioned at four predetermined coordinates to give counts from two central (behind the equator) and two peripheral (in front of the equator) coordinates as previously described [25]. These four images for each eye were then imported into ImageJ (v 1.37), converted to grayscale, and inverted, and the schisis areas were measured by saturating the black areas. For the multiple-injection study, the same four 200 μm areas were used to count the outer nuclear layer cell number. The sample size was six for all the cohorts for single- and multiple-injection studies.

ELISA

Retinal lysates were analyzed with ELISA to detect RS1 expression following the previously described protocol [16,26]. Briefly, isolated neurosensory retinas from mouse eyes were collected and homogenized with a 25G needle using 200 μl cold PBS. Homogenates were then centrifuged for 10 min at 13,000 ×g, and the protein concentration was then determined with the Bradford (Bio-Rad Protein Assay) method in the cleared supernatants. RS1 concentrations were measured using a sandwich ELISA (USCN Life Science Inc.) according to the manufacturer’s instruction [14] and visualized as described for MSCs. All samples were analyzed in duplicate, and the results were calculated as pg/100 mg. For all data, the sample size was three.

Immunohistochemistry

Eyes were enucleated from three mice cohorts (constitutive MSCs, inducible MSCs, and untreated) for single and multiple injections, fixed in 4% paraformaldehyde (PFA, EMS, Hatfield, PA) overnight, and subsequently incubated in 25% (W/V) sucrose for 24 h. The eyes were embedded in Tissue-Tek Cryomolds (Sakura Finetek USA Inc., Torrance, CA) with PolyFreeze Tissue Freezing Medium (Polysciences, Inc., Warrington, PA). Ten-micron-thick sections were cut using a Microm HM 525 microtome (Thermo Fisher, Waltham, MA). Sections were stained to locate MSCs and track RS1 expression. Although tracking MSCs in vivo has been a challenge [27], recent studies showed that PDGF receptor alpha (PDGFRα) can be used for in vitro and in vivo tracking [28]. Immunostaining was performed following established protocols [25]. Briefly, the cryosections were blocked with PBS containing 0.2% Triton X-100 (PBS-T) and 10% (v/v) goat serum for 1 h and labeled overnight with a primary rabbit polyclonal RS1 antibody (1:1,000; gift from Dr. Molday, UBC) and a polyclonal goat PDGFRα antibody (1:50; R&D, Minneapolis, MN). The samples were then rinsed in PBS and labeled for 1 h with secondary antibodies: a goat anti-rabbit immunoglobulin (IgG) conjugated to Alexa Fluor 594 (Life Technologies) for the polyclonal RS1 antibody and donkey anti-goat IgG conjugated to DyLight 488 (Thermo Fisher) for the polyclonal PDGFRα antibody.

Statistics

The significance of the differences between the groups was assessed by performing ANOVA followed by Tukey’s test. A p value of less than 0.05 was considered statistically significant. Data are presented as the mean ± standard error of the mean (SEM).

Characterization of transgene expression by MSCs

RT–PCR demonstrated RS1 expression in constitutive MSCs but not in untransfected AMSCs (Figure 1A). Similarly in inducible MSC RS1 expression was present after induction with 100 ng/ml doxycycline (Figure 1B). Western blotting showed the presence of a 24 kDa RS1 protein in constitutive MSCs (Figure 1C) and after induction by doxycycline in the inducible MSCs (Figure 1D), as seen in the control Weri retinoblastoma cells. Using immunohistochemistry, the RS1 protein was localized to the cytoplasm in the constitutive MSCs and the inducible MSCs (Figure 1E). The amount of RS1 that was secreted into the extracellular media was compared to intracellular RS1 expression (Figure 1F). The constitutive and inducible MSCs secreted about the same amount of RS1 (about 8 ng/million cells/day), and they had similar intracellular amounts of RS1 (13.5 ng/million cells and 12 ng/million cells, respectively).

Topical doxycycline penetration to the posterior pole of the eye

To determine the optimal concentration of topical doxycycline for penetration into the vitreous cavity and the retina for in vivo studies, two different doses of doxycycline were tested (5 and 10 mg/ml). ELISA of the retinal lysates revealed that both doses of doxycycline gave the same lysate doxycycline concentrations of 3 ng/ml (Figure 2A). Therefore, we selected the lower dose of 5 mg/ml for the in vivo studies.

Single injection of RS1-expressing MSCs

After a single injection of either RS1-expressing constitutive MSCs or inducible MSCs into the vitreous cavity of the Rs1h KO mice, the amount of the RS1 protein in the retina was quantitated at 2, 4, or 8 weeks post-injection. No RS1 protein is present in the Rs1h KO mouse retina as previously reported (data not shown) [6]. The constitutive and inducible MSC treatments resulted in 10–12 pg/mg of the RS1 protein in retinal lysates at 2 weeks post-injection (Figure 2B). A gradual decline in RS1 expression was documented over time in both treatment groups. As expected, this decline correlated with previous observations that MSCs do not survive in the retinal microenvironment for more than 6–8 weeks [16].

The localization of RS1-expressing MSCs in the retina was detected using double immunolabeling with antibodies to PDGFRα (28) and RS1 (one) in the treated (constitutive MSCs and inducible MSCs) and untreated Rs1h eyes. Initially, 24 h post-injection, most of the MSCs were in the vitreous, with a few found on the internal limiting membrane (data not shown). At 8 weeks post-injection, RS1-expressing MSCs were detected in the inner nuclear layer (INL) of the retina with a gradient of RS1 staining (Figure 3A). Further away from the MSCs, the surrounding Rs1h retinal cells remained RS1-negative as would be expected compared to the wild-type cells, which showed high levels of RS1 expression in the inner segments and the INL of the retina (Figure 3B).

To determine whether injection of RS1-expressing MSCs had any structural effect on the Rs1h retina, histological sections were assessed. At 8 weeks post-injection, the INL appeared to be intact (Figure 3C) and comparable with the healthy mouse retina (Figure 3B) and differed from that in the untreated control group where schisis cavities were present in the INL (Figure 3C). To quantify the reduction in the schisis cavities, spidergrams were used to plot the size of the cavities in four quadrants of the retina (see the Methods section). The largest cavities were observed at the 2 week post-injection time point in all four quadrants (Figure 4A). Treated animals exhibited smaller schisis cavities in all four quadrants compared to the control groups. This was particularly evident in animals treated with inducible MSCs (Figure 4A). Smaller schisis cavities were also seen in eyes treated with genetically modified MSCs at later time points (Figures 4B,C). To assess the statistical significance of the differences in the schisis cavities seen in the different groups, the total schisis area from all four quadrants was compared at each time point. At 2 weeks post-injection, animals treated with either constitutive MSCs or inducible MSCs showed significantly smaller schisis areas (decreased by 65% and 83%, respectively) compared to the untreated (p<0.0001 for both) and vehicle groups (p<0.001 and p<0.01, respectively; Figure 4D). Similarly, at 4 weeks post injection, the constitutive MSC– and inducible MSC–treated groups had significantly smaller schisis areas than the untreated groups (p<0.0001 and p<0.01, respectively) and the vehicle groups (p<0.01 and p<0.05, respectively). At the 8-week time point, the untreated group had a significantly larger schisis area than the inducible MSC– and constitutive MSC–treated groups (p<0.01 and p<0.05, respectively). We also noted that delivery of unmodified AMSCs had some benefit on the extent of the schisis cavities although this did not reach statistical significance.

To determine whether the histological rescue we observed in the retina translated into functional benefits, ERG and OKT were performed. When measuring the scotopic b-/a-wave ratios of the ERG, a higher ratio indicates a better functional response. At all time points, the treated groups showed a higher ratio than the control groups (Figure 5A). At 4 weeks, the inducible MSC–treated group showed a significantly higher ratio than the untreated control group (2.6 versus 1.4, p<0.05). At the 8-week time point, the ratio for the eyes treated with inducible MSCs was significantly higher than that for all the other groups (p<0.01). These data suggest that the inducible RS1-expressing MSCs resulted in a better ERG response. The visual behavioral responses showed that at all time points the inducible MSC–treated animals demonstrated a higher spatial frequency threshold with a significant difference (p<0.001) at the 4-week time points, compared to the untreated mice (Figure 5B).

Multiple injections of RS1-expressing MSCs

After three injections of either RS1-expressing constitutive MSCs or inducible MSCs into the vitreous cavity of Rs1h mice, the amount of RS1 protein in the retina was quantitated at P120. The constitutive MSC and inducible MSC treatments resulted in approximately 7 pg/100 mg RS1 protein in retinal lysates (Figure 6A). This was two- to threefold higher than with the single injections of cells (2 pg/100 mg RS1 with constitutive MSCs and 3.75 pg/100 mg RS1 with inducible MSCs) at P77 (8 weeks; Figure 2B).

Immunohistochemistry revealed the presence of RS1-expressing MSCs in the inner retina of the treated mice compared to absence of the RS1 protein in the untreated control group (Figure 6B). In addition, the presence of schisis cavities appeared to be reduced (Figure 6C). Quantitation of the size of the schisis cavities was performed at P120 over an 800 µm area in the different treatment groups. Eyes treated with either constitutive MSCs or inducible MSCs had significantly smaller schisis areas (78% reduction) compared to the untreated control group (p<0.01, p<0.05, respectively; n = 6); however, there was no difference between the two treatment groups (Figure 7A). To assess the effect of RS1-expressing MSCs on the preservation of photoreceptor cells, the number of nuclei in the outer nuclear layer (ONL) over an 800 µm area was counted. There were significantly more nuclei in the constitutive MSC–treated group (62%, p<0.0001) and in the inducible MSC–treated group (87%, p<0.0001) compared to the control group (Figure 7B).

As it has been previously reported that the a- and b-waves of the scotopic ERG are decreased after P90 in the Rs1h mouse [26], both components were measured in the different treatment groups (Figure 7C). Treatment with either constitutive MSCs or inducible MSCs resulted in significantly increased a- and b-waves compared to the untreated control group (p<0.05, n = 6). Interestingly, the b-wave in the constitutive MSC treatment group was more than threefold higher than in the control group. This observation was also reflected in the optokinetic tracking responses that showed that constitutive MSC–treated animals had a significantly better spatial frequency threshold (p<0.01, n = 6) compared to the untreated control group (Figure 7D).