Gene therapy has been used experimentally in orthopedics for fracture repair,1–5 vertebral column fusion,6–10 treatment of arthritis,11–18 and, in horses, for treatment of osteoarthritis.19 Introduction of a gene into resident cells to elicit local expression of therapeutic proteins can be more beneficial than direct injection of a recombinant protein into a target tissue because gene therapy may be more cost effective and may yield sustained and higher local tissue concentrations of the desired therapeutic molecules.5,10,18 Administration of desirable genes can be mediated by a number of vector systems, one of which is a replication-deficient adenovirus derived from human adenovirus serotype 5. Recombinant adenoviral vectors are attractive gene transfer vehicles because of their remarkable transduction efficiency, the relative ease of preparing high titers of purified virus, and the ability of adenovirus to infect dividing and nondividing cells.3,5,10,11,18 The fact that genes transferred by adenovirus are expressed for weeks,11,14,20,21 compared with the persistence for years of genes transferred by certain other viral vectors,22–24 makes use of adenovirus vectors appropriate because healing of musculoskeletal tissues can be augmented without persistence of the genes.
Adenovirus vector has been used in horses for gene delivery to cells in vitro and in tissue cultures16,25 as well as to targeted areas in vivo.19 Key processes necessary for adenoviral entry into target cells are the initial recognition of coxsackievirus receptor and subsequent internalization via binding of the arg-gly-asp sequence on the penton base to αvβ5 and αvβ3 integrins.26–28 One of the factors that determines cell permissiveness to adenovirus vector is the number of available receptors on the cell surface, and binding of virus to receptor is the initial requisite step in the infection process. Transduction efficiency can be variable and may be limited in certain cells because of low receptor numbers.26–30 Adenovirus vector may infect even nonpermissive cells when applied at very high MOIs (ie, the number of infectious viral particles per cell), but exposure to high doses of virus may be associated with severe manifestations of cytotoxicity.31,32 Therefore, it is relevant to evaluate cell types with regard to species and tissue of origin to determine their permissiveness and sensitivity to cytotoxic effects resulting from infection with adenovirus vectors. Chondrocytes and synovial cells are the targeted cell types for local gene therapy in joints affected by osteoarthritis or rheumatoid arthritis. Although gene transduction by adenovirus vector in vivo has been reported16,25 in equine chondrocytes and synovial cells, the permissiveness of these articular cell types to adenoviral infection has not been reported.
Stem cells are presently an important focus of investigations of cell-based molecular therapy and tissue engineering.33–35 Although stem cells from adults have a more limited range of differentiation lineages than embryonic stem cells, adult cells can more easily be directed to specific tissue types and are considered safer for transplantation because they have a lower capacity for proliferation and tumorigenicity.33 Bone marrow–derived mesenchymal stem cells are pluripotent and may differentiate into many mesenchymal lineages, including muscle, bone, cartilage, tendon, and ligament, and may be carrier cells for ex vivo gene transfer.34–39 Localized delivery of cells containing transduced genes from adenovirus vector can induce effective tissue concentrations of desired therapeutic proteins and reduce the host immune response such that repeated application of gene therapy is feasible.9,34,40–45 Because BMD-MSCs can be obtained from horses via relatively simple techniques,46 gene therapies may be used for in vivo or ex vivo applications.
Various modifications of adenovirus vectors have been made to increase the efficiency of transduction in certain cell types with low permissiveness to virus vectors, including alteration of receptor binding via fiber pseudotyping,47 use of bifunctional antibodies to connect adenoviral vector to receptors other than coxsackievirus receptor,48 and insertion of heterologous sequences into the fiber knob.49,50 Insertion of the arggly-asp motif into the HI loop of the adenoviral fiber (ie, the beta-sheets between fiber knob H and I proteins) enables the vectors to use the αvβ3 or αvβ5 integrin as an alternative receptor, resulting in increased transduction efficiency in cells deficient in coxsackievirus receptors. Use of this strategy has enhanced the infectivity of virus vector in human tumor cells.50 We selected this arg-glyasp motif–containing adenoviral vectora to investigate whether application of a low titer of adenovirus vector would result in successful gene transfer into equine cells. If successful, it is possible that an efficient therapeutic response could be achieved with minimal adverse effects on surrounding bone or articular structures and the risk of viral distribution into other organs would also be minimized.
Our long-term goal was to determine the permissiveness of various cell types to adenovirus vector–mediated gene transduction and evaluate the efficiency of modified adenoviral vectors for delivering genes to poorly permissive cell types. Transduction efficiency in cells may be enhanced by modification of the adenovirus vector or by selection of different vectors for given types of articular cells if the relative degrees of permissiveness among cell types can be established. Cytotoxic effects in equine cells infected with various doses of adenovirus vector have not previously been compared. Elucidation of these relationships would facilitate the optimal selection of vector vehicle and dose for use in given types of equine cells.
The objective of the present study was to compare permissiveness to adenovirus vector infection and cytotoxic effects of infection with 2 modified serotype 5 adenoviral vectors (E [early region]-1–deleted adenovirus and E-1A–defective adenovirus with a modified capsid containing an arg-gly-asp peptide insert [Ad-RGD]) in equine chondrocytes, synovial cells, and BMD-MSCs. Our hypotheses were that the efficiency of gene transduction would vary among the 3 cell types; high viral titers would induce cytotoxic effects and diminish cell viability; and use of the adenovirus-RGD vector would increase transduction efficiency, particularly in cell types with low permissiveness to vectored gene delivery.
Materials and Methods
Study design—Chondrocytes, synovial cells, and BMD-MSCs were harvested from 15 horses (5 horses were used to obtain each cell type) and cultured in monolayer in duplicate. Human malignant cervical carcinoma of Henrietta Lacksb cells were used as a referent cell type. Permissiveness of each cell type to adenoviral vectors (ie, Ad and Ad-RGD) encoding the marker genes of β-galactosidase (Ad-LacZ) or green fluorescent protein (Ad-GFP and Ad-RGD-GFP) was compared by quantifying transduction efficiency (the percentage of cells expressing the gene) 2 and 7 days after infection. Cytotoxicity of the adenoviral vector in each cell type was evaluated by exposing cells to the adenovirus vector at 6 MOIs (range, 0 to 100), scoring cells histopathologically for morphologic features, staining with trypan blue for cell viability, and conducting cell counts with a hemocytometer.
Horses—The 15 horses included in the study were determined to be clinically normal on the basis of physical and lameness examinations. Horses' median age was 5 years (range, 2 to 15 years). The 10 horses from which articular cartilage (n = 5) and synovium (5) were obtained had tarsocrural joints that were visually, palpably, and radiographically normal prior to euthanasia and joint tissues that were grossly normal at necropsy. Horses from which bone marrow (n = 5) was obtained were clinically normal on physical examination and hematologic evaluation and had visibly and palpably normal sternebrae.
Tissue harvest and cell preparation—Articular cartilage and synovium were harvested from the tarsocrural joint. After aseptic preparation of the skin over the joint, articular cartilage was harvested as 1- to 2-mm full-thickness slices from the trochlear ridges of the talus. Villus synovium was harvested and dissected from underlying adventitia, fat, and joint capsule by use of a dissecting microscope. Chondrocytes and synovial cells were isolated by means of tissue digestion with collagenase and cultured in a monolayer according to a described method.51 Bone marrow–derived mesenchymal stem cells were obtained via bone marrow aspiration from the sternum. Briefly, horses were positioned in dorsal recumbency immediately after euthanasia, the skin was aseptically prepared, and the skin and pectoral muscles were dissected so that the ventral aspect of the sternum was exposed. A bone marrow aspiration needlec was inserted into a vertebral body from the ventrolateral or ventromedial aspect of the sternum, and marrow was aspirated into a sterile, heparin-flushed,d 12-mL syringe. The procedure was repeated until a minimum of 10 mL of bone marrow was collected. Primary BMD-MSCs were isolated via centrifugation of marrow specimens and cultured in a monolayer, as has been described.46 Derived BMD-MSCs were confirmed as pluripotent by culturing in controlled osteogenic, chondrogenic, and adipogenic media containing dexamethasone with ascorbate, recombinant human transforming growth factor-β1, and dexamethasone with insulin and indomethacin, respectively.e Human carcinoma cells (ie, HeLa cells) were chosen as the referent cell type and were purchased.b All cells were cultured in DMEMe supplemented with L-glutamine (300 μg/mL), penicillin (30 μg/mL), streptomycin (30 μg/mL), and 10% fetal bovine serum and incubated at 37°C in a 5% CO2 atmosphere.
Generation of adenoviral vectors—Three types of adenoviral vectors were generated37: a recombinant, replication-deficient, E-1–deleted adenovirus encoding cDNA of β-galactosidase (ie, Ad-LacZ); a recombinant, replication-deficient, E-1A–defective adenovirus encoding cDNA of green fluorescent protein (ie, Ad-GFP); and a recombinant, replication-deficient, E-1A–defective adenovirus encoding complementary DNA of green fluorescent protein as well as containing an arg-gly-asp peptide in the HI loop of the fiber knob domain (ie, Ad-RGD-GFP). Briefly, the cDNA of β-galactosidase or GFP was subcloned into an expression vector and cotransfected with a plasmid containing the 9 to 36 map units of E1-defective human adenovirus 5 into human embryonic kidney 293b cells. The resulting adenoviral vectors were designated Ad-LacZ and Ad-GFP, respectively. Another plasmid containing a gene for fluorescent green protein and additional arg-gly-asp sequences in the HI loop was constructed by means of homologous recombination and was similarly transfected into human embryonic kidney 293 cells. The resulting adenoviral vector was designated Ad-RGD-GFP.a All 3 adenoviral vectors were amplified in human embryonic kidney 293 cells and purified by 3 rounds of centrifugation in cesium chloridef after salt removal via dialysis in a sucrose-containing buffer solution. The number of infectious units of the adenoviral vectors was determined by use of a commercially available kit.g All adenoviral vector preparations were stored at −80°C in Gey's balanced salt solution.f
Cell permissiveness to adenoviral vector infection— The 3 types of equine cells and HeLa cells were cultured until they expanded to confluence and, at a low passage number (2 to 5 passages in equine cells and 20 to 22 passages in HeLa cells), a final cell suspension was placed in 48-well platesh at a density of 10,000 cells/well. Twenty-four hours after the final seeding (day 0), the DMEM was changed to contain Ad-LacZ at concentrations of 0 (control), 1, 5, 10, 50, and 100 MOIs (ie, infectious particles)/cell. At each MOI, equine cells that were seeded in duplicate wells for each of the 5 horses and HeLa cells that were seeded in 5 replicate wells were infected. The volume of DMEM was 500 μL for all wells. In the wells containing 100 MOIs, for example, the 500 μL of DMEM contained 1 million units of Ad-LacZ (ie, 100 U/cell × 10,000 cells/well). Final seeding and application of viral vector were performed at the same time for all cell types, and adenoviral vectors were thawed just before infection.
Transduced cells were quantified at days 2 and 7 after infection by use of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside stainingg and were expressed as the percentage of the total number of cells counted in 5 hemacytomoter fields of 25 × 25-μm area under 200X magnification. If the cell population was low in certain groups, more than 5 fields were used so that a minimum of 1,000 cells/well was counted to ensure accurate calculation. Numbers of equine cells in the duplicate wells were averaged.
Evaluation of adenoviral cytotoxicity in host cells—At days 2 and 7 after infection with adenoviral vector, morphology scores were assigned for cells in each culture well during examination at 200X magnification according to the following scheme: 0 = < 10% of cells had a rounded appearance with minimum signs of cell detachment from the bottom surface of the culture wells; 1 = 11% to 25% of cells had a rounded appearance and had mild signs of cell detachment; 2 = 26% to 50% of cells had a rounded appearance and had moderate signs of cell detachment; 3 = 51% to 75% of cells had a rounded appearance and had severe signs of cell detachment; 4 = 76% to 100% of cells had a rounded appearance, and the cell population was disrupted by severe cell detachment. The median score of cells in 3 representative fields was used for each culture well.
At days 2 and 7, cells in monolayer cultures were harvested by removing media from the well and adding 125 μL of EDTA-trypsinf to each well. After 5 minutes of incubation at room temperature (21°C), the trypsin was neutralized by addition of 125 μL of DMEM to each well; cells were harvested by means of gentle pipetting (250-μL total volume). Ten microliters of harvested cells was mixed with 90 μL of trypan blue solution (10X staining dilution) and placed in duplicate in a hemacytometer.i After 1 minute of incubation at room temperature, total cell numbers and dead cell numbers were counted in 18 of the 1 × 1-mm fields (0.1 mm in depth) in the hemacytometer at 40X magnification. The number of cells per well was calculated as follows: number of cells per well = mean cell counts in the 18 hemacytometer fields (= cell number per 0.1 μL) × 10 (= 100:10-μL staining dilution) × 2,500 (= 250/0.1-μL volume ratio). If cell numbers were low in certain groups, the 2X (100:50 μL) or 1.11X (100:90 μL) staining dilutions and 5 minutes of incubation were used so that a minimum of 30 cells/18 hematocytometer fields were counted for accurate calculation. Cell viability was calculated as the number of dead cells divided by the total cell number in all 18 hemacytometer fields and expressed as a percentage.
Evaluation of sustained transgene expression—The number of transduced cells in each culture well was estimated as follows: number of transduced cells per well = (number of cells per well) × (% viable cells) × (% transduced cells). The calculation was made for each horse by use of the percentage viability and percentage of transduced cells in each MOI group on days 2 and 7.
Comparison of adenoviral vectors with and without the RGD peptide insertion—Equine cells and HeLa cells were seeded in separate 48-well plates at a concentration of 10,000 cells/well. Twenty-four hours after the final seeding (day 0), the medium was changed by adding 500 μL of DMEM containing AdGFP or Ad-RGD-GFP at 0 (control), 1, 5, 10, 50, and 100 MOIs in a volume-controlled manner, as described. The percentage of transduced cells was assessed by use of a fluorescent microscope at days 2 and 7 by calculating the number of cells containing GFP divided by the total number of cells counted within the 5 fields of 25 × 25-μm area under 200X magnification and expressed as a percentage.
Statistical analysis—Data were analyzed with a statistical software program.j Values of P < 0.05 were considered significant. The distribution of objective data (continuous variables) was assessed by use of a subset of normality tests (eg, the ShapiroWilk, Kolmogorov-Smirnov, Cramer-von Mises, and Anderson-Darling tests); results indicated that objective data were normally distributed (P > 0.15). Objective data were analyzed by use of multivariate ANOVA with cell types, MOI group, time points, horse, and 2 types of adenoviral vectors serving as explanatory variables. Horse was treated as a random variable, and the repeated measures were considered nested within time points and horse. Horse was not a significant explanatory variable for all analyses. For the explanatory variables with significant overall P values, multiple subsequent comparisons were made among 4 cell types, among 6 MOI groups, between days 2 and 7, and between the 2 types of adenoviral vectors (Ad-GFP and Ad-RGD-GFP). The subjective cell morphology scores data (categoric variables) were analyzed by use of multivariate ANOVA with cell types, MOI groups, time points, and horse serving as explanatory variables. For explanatory variables with significant Pvalues, subsequent multiple comparisons were similarly made among 4 cell types, among 6 MOI groups, and between days 2 and 7. To clarify the relation between the 2 parts of the experiments, the percentage of transduced cells as calculated by use of Ad-LacZ and Ad-GFP were also compared by use of multivariate ANOVA with cell types, MOI groups, horse, and adenoviral vector types serving as explanatory variables.
Results
Cell permissiveness to adenoviral vector infection—Transduction efficiency was significantly (P < 0.01) higher in HeLa cells than in the equine cells (Figures 1–3). Transduction in BMD-MSCs was significantly (P < 0.01) more efficient than in chondrocytes and synovial cells, and chondrocytes had significantly (P < 0.01) greater transduction efficiency than synovial cells. Therefore, the order of tropism of adenoviral vector for those cell types was HeLa > equine BMD-MSCs > equine chondrocytes > equine synovial cells. For all 4 cell types, the percentage of transduced cells decreased 2- to 7-fold (P < 0.01) between days 2 and 7 in most MOI groups (eg, from 83% to 11% in chondrocytes at 100 MOIs). Transduction efficiency of the Ad-LacZ and Ad-GFP vectors was not significantly different in any cell types in any MOI groups at either time point.
Mean ± SD percentage of transduced cells in 3 types of equine cells and HeLa cells on days 2 (A) and 7 (B) after infection with adenovirus vector. Different letters indicate significant (P < 0.05) differences among values for the 4 cell types at each MOI. Different symbols indicate significant (P < 0.05) differences among values for the 5 MOI groups.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1145
Photomicrographic images illustrating the results of staining on day 2 after infection with Ad-LacZ vector in HeLa cells and equine chondrocytes, synovial cells, and BMD-MSCs. Results for infection of host cells with 1, 10, and 100 MOIs are indicated. Notice that the frequency of transduced cells (blue-staining cells) decreases in order from HeLa cells > BMD-MSCs > chondrocytes > synovial cells and also decreases with lower MOIs.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1145
Photomicrographic images illustrating the results of staining on day 7 after infection with Ad-LacZ vector in HeLa cells and equine chondrocytes, synovial cells, and BMD-MSCs. Results for infection of host cells with 1, 10, and 100 MOIs are indicated. Notice that the frequency of transduced cells (blue-staining cells) decreased in order from HeLa cells > BMD-MSCs > chondrocytes > synovial cells and also decreased with lower MOIs. The frequency of transduced cells was lower on day 7 than on day 2 in all cell types, and severe cytotoxic changes were evident in HeLa cells at 100 MOIs.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1145
Adenoviral cytotoxicity in host cells—The HeLa cells were significantly more sensitive to adenoviral vector dose than any of the 3 equine cell types. For HeLa cells, the number of cells per well was significantly lower than that of any of the 3 types of equine cells (P< 0.01) at both time points and significantly (P< 0.01) lower, even at only 1 MOI, than controls (0 MOI) on days 2 and 7 (Table 1). For all 3 types of equine cells, the number of cells per well was not significantly lower than controls in any MOI groups at day 2 or 7, except for synovial cells in the 100-MOI group on day 2. Overall, the number of cells per well was significantly (P < 0.01) higher on day 7 than on day 2, as a result of cell proliferation (Figures 2 and 3).
Summary of findings pertaining to cytotoxic effects of adenovirus vector (Ad-LacZ) infection at various MOIs in HeLa cells and equine BMD-MSCs, chondrocytes, and synovial cells.
| Cytotoxicity variable | MOIs | |||||
|---|---|---|---|---|---|---|
| 0 | 1 | 5 | 10 | 50 | 100 | |
| Total cell No. (× 103; mean ± SD) | ||||||
| Day 2 HeLa cells | 105 ± 6 | 91 ± 5* | 88 ± 8* | 71 ± 11* | 61 ± 7* | 56 ± 7* |
| BMD-MSCs | 23 ± 4 | 24 ± 1 | 22 ± 4 | 19 ± 2 | 22 ± 1 | 17 ± 3 |
| Chondrocytes | 95 ± 7 | 93 ± 4 | 95 ± 11 | 92 ± 3 | 81 ± 5 | 83 ± 4 |
| Synovial cells | 58 ± 7 | 56 ± 6 | 51 ± 5 | 44 ± 8 | 43 ± 9 | 38 ± 8* |
| Day 7 HeLa cells | 722 ± 86 | 528 ± 49* | 328 ± 96* | 141 ± 74* | 5 ± 2* | 6 ± 3* |
| BMD-MSCs | 117 ± 25 | 116 ± 49 | 116 ± 45 | 121 ± 45 | 105 ± 59 | 87 ± 32 |
| Chondrocytes | 135 ± 36 | 116 ± 39 | 134 ± 55 | 124 ± 42 | 131 ± 82 | 118 ± 49 |
| Synovial cells | 80 ± 43 | 77 ± 29 | 87 ± 23 | 82 ± 22 | 94 ± 42 | 79 ± 42 |
| Cell viability (%; mean ± SD) | ||||||
| Day 2 HeLa cells | 95.7 ± 1.5 | 90.2 ± 2.1* | 85.2 ± 2.4* | 79.6 ± 4.3* | 79.8 ± 2.0* | 72.5 ± 7.8* |
| BMD-MSCs | 97.0 ± 1.3 | 96.9 ± 0.7 | 95.4 ± 2.5 | 96.6 ± 1.7 | 94.2 ± 1.9 | 94.9 ± 1.8 |
| Chondrocytes | 98.2 ± 1.2 | 96.6 ± 1.2 | 96.9 ± 0.8 | 96.8 ± 0.4 | 96.5 ± 0.6 | 96.6 ± 0.9 |
| Synovial cells | 98.4 ± 1.4 | 97.6 ± 1.6 | 97.6 ± 1.4 | 95.4 ± 1.5 | 96.7 ± 1.6 | 96.1 ± 2.4 |
| Day 7 HeLa cells | 98.5 ± 0.2 | 94.5 ± 0.5* | 89.6 ± 0.8* | 76.6 ± 5.2* | 55.8 ± 5.2* | 64.2 ± 5.3* |
| BMD-MSCs | 98.3 ± 0.4 | 97.2 ± 0.8 | 97.5 ± 0.5 | 97.0 ± 0.4 | 97.7 ± 1.0 | 96.8 ± 0.9 |
| Chondrocytes | 98.4 ± 1.3 | 98.5 ± 1.2 | 98.3 ± 1.3 | 98.2 ± 1.1 | 98.8 ± 1.2 | 98.0 ± 1.4 |
| Synovial cells | 98.5 ± 1.3 | 98.5 ± 1.2 | 98.8 ± 0.6 | 97.5 ± 1.8 | 97.4 ± 1.6 | 96.1 ± 2.0 |
| Morphology scores (median [range]) | ||||||
| Day 2 HeLa cells | 0 (0) | 0 (0–1) | 1 (0–1) | 1 (1–2) | 2 (1–2) | 2 (2–3)* |
| All 3 equine cell types | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| Day 7 HeLa cells | 0 (0) | 0 (0–1) | 1 (0–1) | 2 (2–3)* | 3 (3–4)* | 3 (3–4)* |
| All 3 equine cell types | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
Values are significantly (P < 0.05) different from those in control wells (0 MOI) for each cell type.
MOI values = Number of infectious viral particles per cell.
The percentage of viable HeLa cells was significantly lower than the percentage of viability in any of the 3 types of equine cells (P < 0.01) and the percentage of viable cells at higher MOIs (P < 0.01), and the percentage viability was lower on day 7 than on day 2 (P < 0.01). The percentage viability for HeLa cells was significantly lower than that of controls (0 MOI), even at only 1 MOI, on both days 2 and 7 (Table 1). For all 3 types of equine cells, the percentages of viable cells were not significantly less than those of controls in any MOI groups on day 2 or 7.
For HeLa cells, cell morphology scores were significantly higher (indicating more cytotoxic changes) than those of all 3 types of equine cells (P < 0.01), at higher MOIs (P< 0.01), and between days 2 and 7 (P< 0.05). Morphology scores for HeLa cells were significantly higher, compared with those of controls, in the 100-MOI group on day 2 and for the 10-, 50-, and 100-MOI groups on day 7 (Table 1). In all 3 types of equine cells, morphologic signs of cytotoxicity were not observed in any MOI groups or at either day 2 or 7.
Sustained transgene expression—For 3 cell types (ie, chondrocytes, synovial cells, and HeLa cells), the number of transduced cells per well decreased significantly (P < 0.01) between days 2 and 7; the decreases were 2- to 10-fold at most MOIs (eg, from 6.6 on day 2 to 1.2 million cells on day 7 in chondrocytes at 100 MOIs; Figure 4). The number of transduced BMD-MSCs was similar on days 2 and 7, whereas there was no significant difference in the number of transduced cells on days 2 and 7 for all MOI groups.
Mean ± SD estimated number of transduced cells per well on days 2 and 7 after infection of host cells with adenovirus vector. *Values differ significantly (P < 0.05) between days 2 and 7 within an MOI.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1145
Comparison of adenoviral vectors with and without RGD peptide insertion—In all 4 cell types, transduction efficiency was not significantly different between Ad-GFP and Ad-RGD-GFP vectors at any MOI (Figures 5 and 6). As was observed with the Ad-LacZ vector, transduction efficiency decreased between days 2 and 7 (P < 0.01) and signs of cytotoxicity were noticed in HeLa cells at high MOIs.
Mean ± SD percentage of transduced cells by type of adenoviral vector on days 2 and 7 after infection and at various MOIs. Notice that there was no significant difference in transduction efficiency between the Ad-GFP and Ad-RGD-GFP vectors on either day in any cell type, although transduction efficiency decreased in all cell types by day 7.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1145
Images obtained by fluorescent microscopy of HeLa cells and equine chondrocytes, synovial cells, and BMD-MSCs on days 2 and 7 after infection with Ad-GFP or Ad-RGD-GFP (at 100 MOIs). For all cell types and on both days, the percentage of transduced cells was not significantly different between the 2 vector types. Bar = 600 μm.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1145
Discussion
To the authors' knowledge, the present study is the first in which significant differences in tropism of adenovirus vectors for various types of equine cells that are relevant to gene therapy in equine orthopedics has been reported. The equine cells were additionally observed to be more resistant to the cytotoxic effects of adenovirus vector than a human cell line, even at high vector doses. On the basis of these findings, adenovirus vector should be effective at transducing equine chondrocytes, synovial cells, and BMD-MSCs, but different efficiencies of transduction according to cell type should be expected, particularly at low MOIs. It is anticipated that synovial cells will be the most difficult in which to induce gene transduction. This finding may be relevant in regard to injection of vector into an intra-articular space or into an intraarticular fracture site or to the use of coculture systems because gene transfer may not be equal across all cell types, an effect that could result in differential tissue expression of therapeutic proteins, particularly at lower MOIs. Additional studies would be necessary to confirm this effect in vivo.
The present study revealed that adenoviral vector was efficient at gene transduction in the 3 types of equine cells evaluated and in HeLa cells, with efficiency approaching 100% in HeLa cells and equine BMD-MSCs, 80% in equine chondrocytes, and 60% in equine synovial cells at the highest MOI. Manifestations of cytotoxicity were not evident in BMD-MSCs or chondrocytes and were negligible in synovial cells. Wells containing synovial cells with 100-MOI adenoviral infections had smaller cell populations, but the decrease in cell viability was not significant. This may indicate that the primary effect of the high dose of adenoviral vector was to interfere with synovial cell proliferation rather than to induce cell death. The HeLa cells had the highest gene transduction efficiency but also had more cell death and CPEs effects than the other types of cells, even at lower MOI (Figures 1–3). The greater cytotoxic effects observed in HeLa cells may be explained by greater viral replication in human cells, compared with equine cell lines. Small numbers of wild-type human adenovirus 5 in viral preparations may replicate more readily in human cells than in equine cells. In additional studies performed to investigate the possible cause of our findings, we confirmed and quantified the presence of wild-type adenovirus 5 in our vector preparations by performing 6 serial passages of cell lysate onto the equine and HeLa cells. Development of microscopic CPEs in cells indicated wild-type virus replication, which would be amplified with each passage because only wild-type adenovirus 5, and not E-1–deficient adenovirus vector, can replicate in any of those cells. Cytopathic effects from replicating virus were observed in HeLa cells, and the number of cells in which CPEs were observed was increased with every passage. Results of a subsequent dilutional study in which CPEs observed in HeLa cells were used to estimate the adenovirus 5 titer indicated that the adenovirus 5 titer was approximately 0.28% of the Ad-LacZ vector titer. Cytopathic effects, and therefore the presence of replicating adenovirus 5, were not observed in equine cells, even after 6 passages. We postulated that human cell lines may naturally contain factors that facilitate human virus replication. The presence of replication-competent virus in recombinant vector preparations cannot be completely eliminated via the adenoviral vector generation process. Although differences were observed between the human and equine cells in development of CPEs as a result of adenoviral vector gene delivery, further study is necessary to elucidate the mechanisms. The potential adverse effects of adenoviral vectors on cells may be relevant in investigating gene therapy applications in humans. The fact that viral replication was not observed in equine cells may indicate that the adenoviral vector can safely be used in horses.
No significant improvement in efficiency of gene transfer associated with use of the RGD-modified adenoviral vector was observed in the present study. This may reflect the relatively high efficiency of the unmodified adenoviral vector at entering the lines of cells studied. Receptor-mediated transfer may have been maximized such that additional binding of the vector to the integrin receptor on the cell surface did not induce a detectable increase in subsequent gene transcription. The small but significant increase in gene transduction in cells exposed to high MOIs of vector may have resulted from vector entry via other receptorindependent entry mechanisms, such as endocytosis, pinocytosis, or phagocytosis.52,53 In such instances, transduction efficiency would not be expected to be altered by binding of integrin receptors to vector with RGD. It is less likely that failure of this vector to improve transduction efficiency was a result of the lack of integrin receptors on the equine chondrocytes and BMD-MSCs.54 It is more likely that the HeLa and equine cells in our study had a higher density of coxsackie adenoviral receptors, the primary adenovirus receptor, on the cell surfaces. Therefore, increased availability of a secondary vector-cell attachment pathway (ie, via the integrin receptor) did not result in any detectable increase in transduction efficiency. Recent studies48,49,55,56 revealed that incorporation of the RGD peptide into adenovirus or adeno-associated virus vectors can augment the efficiency of transgene expression in certain types of cells that have low expression of coxsackie adenovirus receptors. In comparison, the adeno-associated virus vector has less infectivity than adenoviral vector in certain articular tissues, and transduction efficiency by the former may be significantly improved by this modification.57,58 Cytotoxicity of the RGD-modified vector was not assessed quantitatively in the present study, but the cytotoxic effects appeared to be similar to those induced by Ad-LacZ. It should be emphasized that the viral MOIs in the present study were determined by calculations from infectious unit assays, not from the more crude method of estimating on the basis of optometric particle counts. For this reason, the transduction efficiencies of Ad-LacZ and Ad-GFP were approximately equal, with no significant difference. Thus, the cytotoxic effects should be comparable between different types of adenoviral vectors because the amount of viral infection was standardized by using the infectious units of the vectors rather than crude estimation with particle counts.
Bone marrow–derived mesenchymal stem cells appear to be excellent candidates for use with adenoviral vector in ex vivo gene therapy in horses. First, results indicated that transduction efficiency was significantly higher in BMD-MSCs after infection by adenoviral vector than in chondrocytes or synovial cells. Second, BMD-MSCs did not appear to be sensitive to cytotoxic effects associated with adenoviral infection and continued expression of transduced genes for at least 7 days. The number of transduced BMD-MSCs was similar on days 2 and 7 despite the fact that the number of cells per well increased, resulting in a decrease in the percentage of transduced cells. Because integration of transduced genes into host genome after delivery by adenoviral vector does not occur, it is presumed that only 1 daughter cell after each cell division contained the initial transfected gene. Hence, our results suggest that BMD-MSCs that were initially transfected by adenoviral vector maintained expression of transgenes for a longer period, compared with other cell types. Ex vivo methods of gene delivery can be used to achieve high transduction efficiency, allow clinicians to select specific cell types as carriers, and increase local tissue cellularity. Ex vivo gene delivery may also constitute a safer method of application because there is no direct contact between the adenoviral vector and host cells. The relative ease of obtaining BMD-MSCs makes them promising for use as gene delivery vehicles in ex vivo gene therapy applications, and the fact that they are osteogenic precursors makes these cells promising candidates for use in bone healing.35 Moreover, BMD-MSCs may further enhance fracture healing via their dual autocrine and paracrine actions because they not only secrete an osteoinductive protein (eg, bone morphogenetic protein) but they can also respond to it.35 These properties may explain the potent effects on bone formation that have been reported2,34,40–42,59,60 in various animal models in association with genetically modified BMD-MSCs.
Equine chondrocytes had greater permissiveness to adenoviral vector infection than did synovial cells in the present study, a response that was similar to responses of these equine cell types to adeno-associated viral vector in an earlier study.54 The lower efficiency of gene transduction in equine synovial cells may be explained by a lower density of cell surface receptors. The primary adenoviral receptor (coxsackie adenoviral receptor) is undetectable in human synovial cells20 and is expressed in low numbers in murine synovial cells.61 Equine chondrocytes can readily be transduced by adenoviral vector in vitro, but the process may be limited in vivo because adenovirus vector is generally not able to penetrate the dense extracellular matrix surrounding articular chondrocytes and modify them sufficiently to induce a clinically relevant response.62,63 Conversely, although synovial cells may be less permissive to infection by adenoviral vector, expression of transgenes sufficient for clinical relevance has been obtained in those cells. Synovial lining has a high surface area and direct contact with the joint space; therefore, therapeutic proteins secreted by the synovium in response to the transduced genes can subsequently diffuse into other intra-articular tissues, such as cartilage.63 Therapeutic genes can be efficiently transduced in synovial cells by adenoviral vector, resulting in a high level of intra-articular transgene expression by arthritic synovium.64–67 In 1 study,68 genetically modified cells that were injected into joints engrafted almost entirely in the synovial lining and subsequently secreted transduced gene products.
The percentage of transduced cells decreased between 2 and 7 days after infection, likely as a result of increasing cell numbers and concurrently decreasing numbers of cells containing the transduced genes. This decrease in transgene expression, however, may not be a limiting factor for in vivo gene therapy applications for musculoskeletal disorders in horses. In an earlier study,69 the efficacy of adenovirus as a vector for genes encoding bone morphogenetic protein was sufficient to accelerate the process of bone healing.69 Adenovirus vectors typically do not integrate transgenes into host DNA; hence, a decrease in transgene expression by host cells is observed over weeks to months.11,14,20,21 In earlier studies,21,k chondrocytes used for ex vivo gene delivery persisted as graft cells at a level of 3% to 8% of resident cells at 4 weeks after infection by vector. Genetically modified chondrocytes transplanted in vivo may continue transgene expression at biologically relevant levels for 2 to 4 weeks.11,14,21,25 Therefore, although only small portions of cells maintain transgene expression, this may be sufficient for production of therapeutic proteins at clinically effective concentrations. The relatively rapid loss of transgene expression that has been reported with adenovirus vector was manifested in the present study as a decrease in the numbers of transduced cells within 7 days of exposure to vector (except equine BMD-MSCs). In the in vitro model used in the present study, this effect reflects a lack of integration of vectored genes with host cell genome, resulting in a less intense host immune reaction directed against the vector. Host-cell expression of genes transduced in vivo by adenovirus vector in horses is anticipated to last for 30 to 60 days.19
In summary, the present study revealed that there are variable degrees of permissiveness of equine cell types to adenovirus vector infection, with differences ranked in the following order from most to least permissive: BMD-MSCs > chondrocytes > synovial cells. Transgene expression persisted for at least 7 days in equine BMD-MSCs but waned more quickly in equine chondrocytes and synovial cells. Cytotoxic effects of adenoviral vector on equine cells were minimal, and exposed cells continued to proliferate between days 2 and 7. Modification of the vector by addition of an RGD peptide did not significantly increase the efficiency of transduction.
ABBREVIATIONS
| BMD-MSCs | Bone marrow–derived mesenchymal stem cell |
| HeLa | Human malignant cervical carcinoma of Henrietta Lacks |
| CPE | Cytopathic effect |
| DMEM | Dulbecco modified Eagle medium |
| Ad-LacZ | Adenovirus encoding β-galactosidase |
| Ad-GFP | Adenovirus encoding green fluores cent protein |
| Ad-RGD-GFP | Adenovirus encoding GFP and an arggly-asp peptide in the fiber knob domain |
| MOI | Multiplicity of infection |
Sidney Kimmel Cancer Center, San Diego, Calif.
American Type Culture Collection, Rockville, Md.
MD Tech Inc, Gainesville, Fla.
American Pharmaceutical Partners Inc, Schaumburg, Ill.
Gibco, Grand Island, NY.
Sigma Chemical Co, St Louis, Mo.
BD Biosciences Clontech, Palo Alto, Calif.
Corning Inc, Corning, NY.
Hausser Scientific, Horsham, Pa.
SAS Institute Inc, Cary, NC.
Goodrich LR. Enhanced early healing of articular cartilage with genetically modified chondrocytes expressing insulin-like growth factor-I (abstr). Vet Surg 2002;31:482.
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