The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 88, Issue 4

Scientific Articles | April 01, 2006

Safety and Efficacy of Ultraviolet-A Light-Activated Gene Transduction for Gene Therapy of Articular Cartilage Defects

Michael D. Maloney, MD¹; J. Jeffrey Goater, MS¹; Richard Parsons, DVM¹; Hiromu Ito, MD, PhD¹; Regis J. O'Keefe, MD, PhD¹; Paul T. Rubery, MD¹; M. Hicham Drissi, PhD¹; Edward M. Schwarz, PhD¹

¹ The Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Box 665, Rochester, NY 14642. E-mail address for E.M. Schwarz: edward_schwarz@urmc.rochester.edu

View Disclosures and Other Information

In support of their research for or preparation of this manuscript, one or more of the authors received grants or outside funding from LAGeT, Inc. (Rochester, New York), which was founded by five of the authors (M.D.M., J.J.G., P.T.R., R.J.O'K., and E.M.S.). None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Note: All of the rAAV preparations were provided by R.J. Samulski and the University of North Carolina-Chapel Hill Vector Core Facility.

Investigation performed at the Center for Musculoskeletal Research, University of Rochester, Rochester, New York

The Journal of Bone and Joint Surgery, Incorporated

J Bone Joint Surg Am, 2006 Apr 01;88(4):753-761. doi: 10.2106/JBJS.E.00400

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Abstract

Background: Gene therapies for articular cartilage defects are limited by the absence of an in vivo delivery system that can mediate site-specific transduction restricted to within the margins of the defect during routine arthroscopy. We have proposed the use of ultraviolet light to stimulate gene expression following infection by recombinant adeno-associated virus (rAAV). However, research has demonstrated that short-wavelength ultraviolet light (ultraviolet C), while effective, is neither safe nor practical for this purpose. We evaluated the safety and efficacy of long-wavelength ultraviolet light (ultraviolet A) from a laser to induce light-activated gene transduction in articular chondrocytes in vitro and in vivo.

Methods: The effects of ultraviolet A from a 325-nm helium-cadmium laser, delivered through a fiberoptic cable, on cytotoxicity, mutagenesis, intracellular reactive oxygen species, and light-activated gene transduction of human articular chondrocytes were evaluated in dose-response experiments of primary cultures. Cytotoxicity was determined by trypan blue exclusion. The presence of pyrimidine dimers in purified genomic DNA was determined by enzyme-linked immunosorbent assays. Intracellular reactive oxygen species levels were determined by flow cytometry at one hour and twenty-four hours. In vitro light-activated gene transduction with rAAV vectors expressing the green fluorescent protein (eGFP) or ß-galactosidase (LacZ) was determined by fluorescence microscopy and bioluminescence assays, respectively. In vivo light-activated gene transduction was quantified by stereotactic immunohistochemistry for ß-galactosidase in rabbit articular cartilage defects in the patellar groove that had been irradiated with ±6000 J/m² of ultraviolet A one week after direct injection of 10⁷ transducing units of rAAV-eGFP.

Results: Ultraviolet A failed to induce significant cytotoxicity at all fluencies below 6000 J/m². Dose-dependent cytotoxicity was observed at greater fluencies. In contrast to ultraviolet C, which induced significant (p < 0.05) pyrimidine dimer formation at all fluencies in a dose-dependent manner, ultraviolet A failed to induce DNA modifications. Conversely, ultraviolet C proved to be a poor inducer of intracellular reactive oxygen species, while ultraviolet A immediately induced high levels of intracellular reactive oxygen species, which were completely resolved twenty-four hours later. Ultraviolet A demonstrated significant light-activated gene transduction effects in vitro, which were dose-dependent (p < 0.05). In vivo, ultraviolet A mediated a tenfold increase in transduction in which 40.8% of the superficial chondrocytes adjacent to the defect stained positive for green fluorescent protein compared with 5.2% in the knees treated with no ultraviolet A (p < 0.006).

Conclusions: These results provide what we believe is the first formal demonstration of an agent that can induce rAAV transduction in the complete absence of cytotoxicity and DNA modification. They also suggest that the mechanism by which long-wavelength ultraviolet light mediates site-specific gene expression is by means of the induction of intracellular reactive oxygen species. Finally, laser-derived ultraviolet A can be readily transferred through a fiberoptic cable to mediate light-activated gene transduction in vivo.

Clinical Relevance: This is the first demonstration of in vivo site-directed gene delivery to articular defects with use of a method that is highly compatible with standard arthroscopy. Future studies with chondrogenic genes and longer outcome measurements are warranted to evaluate the potential of this gene therapy approach for superficial articular defects and meniscal tears.

Figures in this Article

It has been estimated that between 5% and 10% of young active patients presenting with a knee hemarthrosis after a specific traumatic event have an articular cartilage injury¹. It is unknown how many of these articular cartilage injuries become symptomatic. However, because of the limited reparative capacity of articular cartilage, the progression of articular degeneration after an injury has been well documented, and articular cartilage defects from an injury are a leading cause of osteoarthritis^2-4. Present surgical treatment options include arthroscopic lavage and débridement, marrow-stimulation techniques, osteochondral autograft transfer, and autologous chondrocyte implantation. To date, there is no treatment that achieves repair of a defect by means of the regeneration of normal, healthy cartilage. To this end, various gene-therapy approaches are currently under investigation^5,6. The goal of this treatment is to mimic the embryonic gene expression that mediates chondrogenesis during development, similar to that induced in animals competent for epimorphic regeneration during blastema formation⁷. While early work in this field has indicated a need to direct the target gene expression to the defect, a safe and effective method to achieve site-specific gene delivery remains elusive. One potential approach to achieve this by means of recombinant adeno-associated virus (rAAV)-mediated gene therapy is to pretreat the target tissue with agents that render the cells competent for immediate second-strand synthesis⁸. These agents, which were originally chosen on the basis of their ability to induce DNA polymerases, simultaneously induce cytotoxic and mutagenic side effects. As an example, short-wavelength ultraviolet light (ultraviolet C) mediates light-activated gene transduction in human articular chondrocytes⁹ and mesenchymal stem cells10 only at fluencies that induce substantial cell death from apoptosis. However, this treatment did not have significant effects on the proliferation, differentiation, and matrix production of the surviving cells. To overcome these obstacles, we tested the hypothesis that long-wavelength ultraviolet light (ultraviolet A) induces site-specific light-activated gene transduction in articular chondrocytes by increasing intracellular reactive oxygen species without damaging DNA.

Materials and Methods

Materials and Methods | Results | Discussion | References

Isolation of Human Articular Chondrocytes and Fibroblast-Like Synoviocytes

Primary human articular chondrocytes and fibroblast-like synoviocytes were isolated as previously described^9,11. Briefly, human articular chondrocytes were isolated from articular cartilage explant samples. The cartilage explant samples were obtained with informed consent, as well as with the approval of the Research Subjects Review Board of the University of Rochester. The explant cartilage was carefully dissected from the subchondral bone. The finely chopped fragments of articular cartilage were washed twice with 5 mL of sterile phosphate-buffered saline solution (pH = 7.4) in a 50-mL conical tube (VWR International, West Chester, Pennsylvania), prior to centrifugation for five minutes at 300 g. The supernatant was removed, and a two-step enzymatic digestion was performed. The first digestion was done with 1% testicular hyaluronidase (Sigma, St. Louis, Missouri) in 1:1 Dulbecco's Modified Eagle Medium (DMEM)/Ham's F12 with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen, Carlsbad, California) for one hour at 37°C. Afterward, the sample was centrifuged for five minutes at 300 g, and the supernatant was discarded. For the second digestion, the sample was then resuspended in 1:1 DMEM/Ham's F12 with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% clostridial collagenase A (Sigma) and was incubated overnight at 37°C. Both incubations were completed with vigorous shaking. After the final digestion, the solution was filtered through a 40-µm cell strainer (BD Biosciences, Franklin Lakes, New Jersey), and the chondrocytes were pelleted by means of centrifugation for five minutes at 300 g. The supernatant was removed, the chondrocytes were resuspended in fresh 1:1 DMEM/Ham's F12 with 10% fetal bovine serum and 1% penicillin-streptomycin, and they were counted, plated, and grown in a 5% CO₂ atmosphere in a water-jacketed incubator (Forma Scientific, Marietta, Ohio) at 37°C.

Irradiation of Cells with Ultraviolet A

Ultraviolet-A irradiation of the cell cultures was performed with use of the 325-nm output of a helium-cadmium laser (Omnichrome 2074 35/100M; Melles Griot, Carlsbad, California). This laser emits approximately 35 mW at 325 nm and 100 mW at 442 nm. To deliver laser light to the surface of an adherent cell sample, the output of the laser was focused into a subminiature version-A-terminated multimode optical fiber with high ultraviolet transmission (FG-550-UER; Thorlabs, Newton, New Jersey). The distal end of the fiber is fitted and fixed at a distance above the sample such that the desired irradiation spot size is achieved. For in vitro studies, the distal end was fixed at 9.5 cm above the plate to irradiate a 2.2-cm-diameter area, which covered all of the cells in the twelve-well plate. For in vivo studies, the distal end was placed approximately 2.5 cm from the defect to irradiate an area that was approximately 5 mm in diameter, which covered all of the cells within 2 mm of the edge of the defect. The intensity exiting the fiber was approximately 12 mW at 325 nm. To achieve accurate dosimetry, a rotary shutter blade (Electro-Optical Products, Glendale, New York) is inserted into the laser beam path between the laser head and the optical fiber coupler. The opening of the shutter is controlled with a manually activated digital timer system. The duration of the laser exposure varied such that the dose ranged from 600 to 15,000 J/m².

Irradiation of Cells with Ultraviolet C

Ultraviolet-C irradiation of the cells was performed as described previously, with use of the Spectrolinker XL-1500 (Spectronic, Leeds, United Kingdom)⁹, which is an enclosed unit that exposes all of the cell evenly from an overhead lamp. The XL-1500 has an output power of 15 W, and the desired energy dose (J/m²) was manually set by means of the digital interface on the device.

Quantification of rAAV Transduction

The treatment of human articular chondrocytes was carried out in ninety-six-well dishes with approximately 6 × 10⁴ human articular chondrocytes plated in each well. The cells were allowed to adhere overnight. Immediately before infection of the cells, the medium was removed from the culture and the cells were irradiated with ultraviolet A or ultraviolet C. The cells were then infected at a multiplicity of infection of 100 with use of an rAAV carrying either enhanced green fluorescent protein (eGFP) or the bacterial ß-galactosidase reporter gene (LacZ), driven by the cytomegalovirus promoter. The rAAV was prepared by the University of North Carolina-Chapel Hill Gene Therapy Vector Core Facility. Afterward, 100 µL of fresh media (1:1 DMEM/Ham's F12 with 10% fetal bovine serum and 1% penicillin-streptomycin) was added to the wells, and the cells were incubated (in 5% CO₂ at 37°C) for two days before harvest for analysis by either fluorescence microscopy¹⁰ or chemiluminescence with use of the Luminescent ß-galactosidase Genetic Reporter System (BD Biosciences, San Jose, California)¹² as we have described previously.

Measurement of Relative Reactive Oxygen Species Levels

Human articular chondrocytes were seeded in twelve-well dishes and treated with ultraviolet light as described above. Immediately after treatment, 1 mL of the appropriate cell culture medium was reintroduced into the wells and the cells were cultured for either one hour or twenty-four hours prior to being harvested. At harvest, the cells were rinsed twice with 500 µL of phosphate-buffered saline solution (pH = 7.4) and resuspended in 500 µL of ice-cold phosphate-buffered saline solution. Then, dichlorofluorescein (DCFH; Invitrogen) was added to the cell mixture at a final concentration of 50 µM. The samples were then incubated in the dark for thirty minutes at 37°C with periodic mixing before analysis with use of a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, California).

Determination of Ultraviolet Cytotoxic Effects

The human articular chondrocytes were treated with escalating doses of ultraviolet A (600 to 15,000 J/m²). Cell death was determined twenty-four hours after ultraviolet treatment by trypan blue staining, as we described previously⁹. A total of 300 cells were counted for each treatment group. Values are expressed as the mean and the standard deviation of the percentage of trypan-positive cells.

Measurement of Relative Pyrimidine Dimer Levels

Genomic DNA was isolated from the human articular chondrocytes immediately after treatment with ultraviolet A (600 to 6000 J/m²) or ultraviolet C (50 to 1000 J/m²) and was used to coat a ninety-six-well enzyme-linked immunosorbent assay (ELISA) plate (150 ng/well). The wells were blocked for two hours with use of phosphate-buffered saline solution containing 10% bovine serum albumin. A thymine dimer-specific antibody conjugated to horseradish peroxidase (Kamiya Biomedical, Seattle, Washington) was used to probe for DNA damage. The ELISA was developed with Substrate A and B (R and D Systems, Minneapolis, Minnesota) according to the manufacturer's instructions. The plate was read with use of an ELISA plate reader (Tecan; Phenix Research Products, Candler, North Carolina). The values are expressed as the mean optical density and the standard deviation.

Effects of Ultraviolet A on Light-Activated Gene Transduction In Vivo

The effects of 325 nm of ultraviolet-A light-activated gene transduction on articular chondrocytes in vivo were evaluated with use of a well-established rabbit model in which an articular defect was made in the patellar trochlea¹³. Two groups of five female New Zealand White rabbits (4.5 to 4.7 kg) were studied following protocol approval by the University of Rochester Committee for Animal Resources. Anesthesia was provided by a single intramuscular injection of a mixture of ketamine (35 mg/kg) and xylazine (5 mg/kg). Afterward, a surgical plane of anesthesia was maintained with isoflurane gas (2% to 2.5%). The defect was generated with a punch after exposing the articular cartilage of the patellar trochlea through an anteromedial parapatellar arthrotomy and lateral luxation of the patella. Then, the defect was exposed to zero or 6000 J/m² of ultraviolet A. Previously, we demonstrated that rAAV infection is very efficient, reaching its peak after only ten minutes of direct interaction between the virus and the cells¹⁰. Thus, to deliver the vector to the chondrocytes at the edge of the defect, 10⁷ transducing units of rAAV-eGFP in 50 µL of phosphate-buffered saline solution was injected through a gas-tight syringe (Sample Lock Syringe; Hamilton, Reno, Nevada) into the defect and allowed to pool there for ten minutes. Afterward, the knee capsule and wound were closed in layers while hemostasis was secured by cauterization. At this time, the excess vector is presumed to be washed away from the defect. For pain management, the rabbits received 1.1 mg/kg of intravenous Banamine (flunixin meglumine) preoperatively, followed by 1.1 mg/kg of Banamine administered intramuscularly or subcutaneously once a day for two days postoperatively if indicated by clinical signs.

The rabbits were killed with use of a lethal dose of pentobarbital one week later. Both the experimental knee and the un-involved, contralateral control knee were harvested, decalcified in EDTA, embedded in paraffin, and sectioned with a microtome into 3-mm sections parallel to the articular surface. Three serial sections were prepared at increments of 200 µm from the articular surface to the osteochondral tidemark for immunohistochemistry with use of antibodies specific for eGFP (Clontech, Mountain View, California) as we described previously⁹. Transduction efficiency was quantified, with use of stereotactic histomorphometry, by three reviewers (M.D.M., P.T.R., and J.J.G.) who were blinded to the treatment. The reviewers counted the total number of articular chondrocytes, the number of eGFP-positive chondrocytes, and the number of empty lacunae in the fields immediately adjacent to the defects (1.3 mm lateral to the defect) in the photomicrographs made at 40× magnification. The mean of the three scores was used as the value for each sample. These five values were then used to calculate the mean and standard deviation for the group.

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Fig. 1: Ultraviolet A-induced cytotoxicity in human articular chondrocytes (AC) and human fibroblast-like synoviocytes (FLS). Human articular chondrocytes and human fibroblast-like synoviocytes were grown in monolayer culture, exposed to the indicated doses of ultraviolet A (UVA), and stained with 0.2% trypan blue twenty-four hours after treatment. The values are presented as the mean percentage (and standard deviation) of trypan blue-positive cells. The results shown represent three independent experiments. The asterisk indicates a significant difference compared with the unexposed control (p < 0.05).

Statistics

Quantitative analyses are presented as the mean and the standard deviation. Analysis of variance (in vitro studies) or the Student t test (in vivo studies) were performed to determine significant differences between groups, with the level of significance set at p < 0.05.

Results

Materials and Methods | Results | Discussion | References

Sensitivity of Human Articular Chondrocytes and Human Fibroblast-Like Synoviocytes to Ultraviolet A

We previously determined^9,12 that human articular chondrocytes and fibroblast-like synoviocytes are highly sensitive to ultraviolet C, which induces significant (p < 0.05) cell death at 100 J/m². To determine the cytotoxic effects of ultraviolet-A treatment on the prominent cells in the joint, we exposed human articular chondrocytes and human fibroblast-like synoviocytes to various doses and assayed for trypan blue exclusion immediately after irradiation and twenty-four hours later. While no significant cell death was seen immediately after irradiation (data not shown), significant cell death was seen at twenty-four hours in cultures treated with 6000 J/m² and higher (p < 0.05) (Fig. 1). The amount of cell death seen on day 2 and day 3 after irradiation did not differ significantly from the levels seen at twenty-four hours (data not shown). Thus, human articular chondrocytes were able to tolerate much higher doses of 325-nm ultraviolet A compared with 254-nm ultraviolet C (LD50 [median lethal dose] = 12,000 compared with 500 J/m²)⁹.

Ultraviolet A Induces rAAV Gene Transduction in Human Articular Chondrocytes

To assess ultraviolet-A light-activated gene transduction in human articular chondrocytes, cultures were exposed to various doses immediately prior to infection with either rAAV-eGFP or rAAV-LacZ. The cells were harvested forty-eight hours later, and transduction efficiency was determined by either fluorescence microscopy for eGFP or ß-galactosidase activity with use of a chemiluminescence assay (Fig. 2). Ultraviolet A caused significant light-activated gene transduction effects in a dose-dependent manner starting at 600 J/m² (p < 0.05). Moreover, in contrast to ultraviolet C, which only mediates light-activated gene transduction in human articular chondrocytes at doses that induce significant cell death⁹, ultraviolet A has a broad range (600 to 6000 J/m²) that can induce light-activated gene transduction without cytotoxic side-effects, as indicated by the results in Figure 1.

DNA Damage Effects of Ultraviolet C and Ultraviolet A in Human Articular Chondrocytes

To determine the relative capacity of ultraviolet C and ultraviolet A to induce DNA damage in the human articular chondrocytes, we utilized a pyrimidine dimer assay. Pyrimidine dimers, more specifically thymine dimers, are the most common form of damaged DNA caused by ultraviolet light and result from the cross-linking of two adjacent thymine bases at the carbon-five and carbon-six positions, respectively¹⁴. To investigate this mutagenesis, human articular chondrocyte cultures were exposed to various doses of either ultraviolet C or ultraviolet A. Genomic DNA was isolated from the cells and probed for pyrimidine dimers with use of a specific ELISA. Ultraviolet-C irradiation at all fluencies tested resulted in significant dimer formation in a dose-dependent fashion (p < 0.05) (Fig. 3). Conversely, ultraviolet A failed to induce pyrimidine dimer formation above background, at any dose tested. In order to exclude the possibility that ultraviolet A induces less common forms of DNA damage, we also looked at the levels of pyrimidine (6-4) pyrimidone photoadducts and abasic sites. The results demonstrated that neither ultraviolet C nor ultraviolet A could induce significant differences compared with untreated controls (data not shown). Taken together, these results provide what we believe is the first formal demonstration that ultraviolet A does not alter DNA biochemistry at doses that augment rAAV transduction.

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Fig. 2: Ultraviolet A-induced rAAV transduction in human articular chondrocytes. Human articular chondrocytes were grown in monolayer culture, were pretreated with the indicated dose of ultraviolet A, and were subsequently infected with rAAV-eGFP (a) or rAAV-LacZ (b). Transduction efficiencies were determined forty-eight hours after infection, as described in the Materials and Methods section. Fluorescent images of representative fields are shown at 40× magnification (a). The ß-gal expression in relative light units (RLU) is presented as the mean and the standard deviation for three independent experiments (b). The asterisk indicates a significant difference compared with rAAV alone (p < 0.05).

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Fig. 3: Ultraviolet-induced pyrimidine dimer formation in human articular chondrocytes. Human articular chondrocytes were grown in monolayer culture and were treated with the indicated doses of either ultraviolet A (UVA) or ultraviolet C (UVC). Immediately after ultraviolet exposure, the cells were harvested, genomic DNA was isolated, and pyrimidine dimer levels were determined by ELISA. The values represent the mean and the standard deviation of the optical density at 595 nm for three independent experiments. The asterisk indicates a significant difference compared with the unexposed control (p < 0.05).

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Fig. 4: Ultraviolet-induced reactive oxygen species levels in human articular chondrocytes. Human articular chondrocytes were grown in monolayer culture and were treated with the indicated doses of either ultraviolet A (UVA) or ultraviolet C (UVC). The cultures were harvested at either one hour or twenty-four hours after treatment and were analyzed for relative reactive oxygen species levels by means of a dichlorofluorescein (DCFH) fluorescence-activated cell sorter. The values in the graph represent the mean percentage (and standard deviation) of the DCFH-stained human articular chondrocytes that have a greater fluorescence intensity than 99% of the unstained human articular chondrocyte controls from three independent experiments. The asterisk indicates a significant difference compared with the unexposed controls (p < 0.05). FL1-H = relative mean fluorescence intensity.

Ultraviolet-A Induction of rAAV Transduction

On the basis of previous studies that investigated the differential cellular effects of ultraviolet A and ultraviolet C^15,16, we hypothesized that the mechanism by which ultraviolet A is able to induce rAAV transduction is through the upregulation of intracellular reactive oxygen species. To investigate, we exposed cultures to various doses of ultraviolet A and ultraviolet C. At one hour and twenty-four hours after irradiation, we analyzed the cultures for reactive oxygen species using the dichlorofluorescein substrate. The results showed that ultraviolet C is a very poor inducer of intracellular reactive oxygen species even at doses that stimulate significant cytotoxicity and pyrimidine dimer formation (Fig. 4), confirming that the central mechanism of ultraviolet C-induced light-activated gene transduction is the cellular response to DNA damage. In contrast, ultraviolet A is a very potent inducer of intracellular reactive oxygen species, as peak levels in 50% to 87% of the human articular chondrocytes were achieved at doses that mediated light-activated gene transduction without cytotoxicity or DNA mutations. Thus, this suggests a unique mechanism for ultraviolet A-induced light-activated gene transduction compared with ultraviolet C and other known DNA-damaging agents. Furthermore, the increase in reactive oxygen species was transient, returning to control levels twenty-four hours after irradiation.

Ultraviolet A-Mediated Light-Activated Gene Transduction In Vivo

In order to demonstrate the safety, efficacy, and utility of ultraviolet A-mediated light-activated gene transduction in vivo, we performed an experiment in which an open articular cartilage defect in the patellar groove of the rabbit was generated, irradiated with zero or 6000 J/m² of ultraviolet A, and infected with 10⁷ transducing units of rAAV-eGFP before closing. The knees were harvested one week later, and the survival and transduction of the articular chondrocytes immediately adjacent to the defect were determined by stereotactic histomorphometry. In terms of the side effects of ultraviolet A, we did not observe any problems with wound-healing, cartilage degradation outside the defect, or significant cell death, as determined by empty lacunae (Table I). However, the effects of ultraviolet A on the transduction of superficial articular chondrocytes were remarkable, as demonstrated in the representative histologic findings from the two groups presented in Figure 5. To quantify the differences in transduction efficiency, we determined the total number of chondrocytes and the number of eGFP-positive chondrocytes to derive the percentage of eGFP-positive articular chondrocytes in the region 1.3 mm lateral to the defect at 40× magnification (see Fig. 5, E and 5, J). This analysis, presented in Table I, revealed that the ultraviolet-A treatment significantly enhanced rAAV transduction tenfold (p < 0.006). In addition to the 0-J/m² negative control, examination of the entire transverse section of the treated distal femoral condyles and the contralateral knees also failed to detect eGFP-positive articular chondrocytes (data not shown), indicating that we had achieved site-specific light-activated gene transduction.

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TABLE I In Vivo Transduction Efficiency and Cytotoxicity of Ultraviolet A-Induced Light-Activated Gene Transduction^*

Ultraviolet A	Total No. of Chondrocytes	No. (%) of eGFP-Positive Chondrocytes	No. (%) of Empty Lacunae
0 J/m²	168.8 ± 11.3	8.8 ± 3.9 (5.2)	3.9 ± 1.2 (2.3)
6000 J/m²	144.7 ± 18.4	59.0 ± 13.0 (40.8)	5.4 ± 0.5 (3.6)
P value†	0.300	0.006	0.288

The values are given as the mean and the standard deviation. eGFP = enhanced green fluorescent proteinStudent t test

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TABLE I In Vivo Transduction Efficiency and Cytotoxicity of Ultraviolet A-Induced Light-Activated Gene Transduction^*

Ultraviolet A	Total No. of Chondrocytes	No. (%) of eGFP-Positive Chondrocytes	No. (%) of Empty Lacunae
0 J/m²	168.8 ± 11.3	8.8 ± 3.9 (5.2)	3.9 ± 1.2 (2.3)
6000 J/m²	144.7 ± 18.4	59.0 ± 13.0 (40.8)	5.4 ± 0.5 (3.6)
P value†	0.300	0.006	0.288

The values are given as the mean and the standard deviation. eGFP = enhanced green fluorescent proteinStudent t test

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Fig. 5: The in vivo effects of ultraviolet A-induced light-activated gene transduction in articular cartilage defects. The rabbits underwent knee surgery that involved the creation of a patellar groove defect, irradiation with zero or 6000 J/m² of ultraviolet A, and delivery of 10⁷ transducing units of rAAV-eGFP. One week later, the knees were harvested for histologic analysis. Sections were stained with hematoxylin and eosin (A and F) or immunostained for eGFP (B through E and G through J), and representative photomicrographs were made of the whole defect (A, B, F, and G) at 4× magnification, of the edges of the defect (C, D, H, and I) at 10× magnification, and of a region of interest 1.3 mm away from the edge of the defect (E and J), which was used for quantification, at 40× magnification. The brownish-red color (B through E and G through J) indicates positive immunostaining for eGFP.

As expected, eGFP transduction of articular chondrocytes had no effect on cartilage-healing, as evidenced by the normal fibrotic response (Fig. 6). However, this finding indicates the lack of side effects on normal healing as a result of the ultraviolet-A and rAAV treatment.

Discussion

Materials and Methods | Results | Discussion | References

After routine arthroscopic surgery to stabilize the margins of superficial tears in articular and meniscal cartilage, a permanent defect in the tissue remains. Evolving techniques have emerged to address these defects. Arthroscopic and staged arthroscopic and open procedures are showing improved symptom resolution, yet reparative tissue remains distinct from the native tissue¹⁷. Since human cartilage is incapable of mounting a true repair response to this injury, the defect remains and is at high risk of osteoarthritic changes. As a potential solution to this problem, investigators have proposed gene therapies that aim to recapitulate chondrogenic gene expression known to mediate cartilage formation during development. This theory is supported by the biology of epimorphic regeneration in lower vertebrates such as amphibians^18-20, which can regrow amputated skeletal elements that include articular cartilage. However, in addition to the specific genes that are expressed in the healing tissue, it has become clear that the temporal and spatial regulation of this gene expression is equally important. Thus, it is unclear how a gene therapy designed to treat a cartilage defect could succeed without inducing a morphogenic gradient at the edge of the defect. Unfortunately, there is no current technology that can mediate site-specific gene delivery to chondrocytes at the edge of a cartilage defect. This includes tissue-specific promoters and pseudotyped viral vectors²¹, which cannot distinguish chondrocytes in immediate proximity to the defect compared with chondrocytes in healthy normal cartilage far away from the defect.

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Fig. 6: Ultraviolet-A and rAAV-eGFP treatment does not affect the normal process of fibrous deposition and healing of the defect at seven days. The accumulation of fibrotic tissue seen in the defect of a rabbit treated with ultraviolet A and rAAV-eGFP from a hematoxylin and eosin-stained section photographed at 4× (left) and 200× (right) magnification was indistinguishable from fibrosis observed in the control group.

On the basis of the original observations that ultraviolet C facilitates rAAV transduction by inducing DNA repair enzymes that mediate second-strand synthesis²², we proposed that light-activated gene transduction could be used for site-directed gene therapy to the edge of articular cartilage defects and demonstrated its efficacy for this purpose⁹. However, intense investigations over the last decade have failed to overcome the serious side effects of ultraviolet C-induced light-activated gene transduction. Furthermore, ultraviolet C has a very unfavorable absorption profile, such that it cannot be readily transmitted to an articular defect during routine arthroscopy. We hypothesized that ultraviolet A would be clinically useful as an alternative to ultraviolet C for light-activated gene transduction because (1) ultraviolet A is not absorbed by DNA and therefore cannot directly induce DNA mutations, (2) it can be readily transmitted through a fiberoptic cable to an articular defect during arthroscopy, and (3) it can be produced by a high-energy laser such that only short exposures (<30 seconds) would be needed to induce light-activated gene transduction.

In this study, we present what we believe is the first evidence that ultraviolet A can induce rAAV transduction without simultaneously causing cytotoxicity and DNA mutations. Moreover, the window between significant light-activated gene transduction effects and significant cytotoxicity appears to be one order of magnitude (>600 to <6000 J/m²). We also demonstrated that this effect is associated with the transient induction of high levels of intracellular reactive oxygen species. In so doing, we identified a novel mechanism to mediate light-activated gene transduction without DNA mutations. In contrast to ultraviolet C, which mediates light-activated gene transduction secondary to DNA mutations, ultraviolet A-induced light-activated gene transduction effects appear to be dependent on transient reactive oxygen species upregulation.

In order to demonstrate the safety, efficacy, and feasibility of ultraviolet A-induced light-activated gene transduction for gene therapy of articular cartilage defects in vivo, we performed a pilot study in rabbits. In the present study, we showed that direct in vivo administration of rAAV is very inefficient for transducing articular chondrocytes, which is consistent with the findings of previous reports on undamaged cartilage^12,23 and a recent study on rAAV transduction in osteochondral defects in which few articular chondrocytes were transduced relative to the bone marrow-derived repair tissue²⁴. Remarkably, pretreatment with 6000 J/m² of ultraviolet A leads to a tenfold increase in the transduction of these articular chondrocytes after one week, as 44.6% of the cells in the superficial layer expressed the transgene. Additionally, this treatment does not lead to increased rAAV transduction in the deeper layers of cartilage, as evidenced by the complete absence of eGFP-positive chondrocytes beneath the intermediate zone in Figure 5, B through 5, E and 5, G through 5, J. This is likely due to the fact that the 26-nm viral particle cannot penetrate the dense extracellular matrix and is restricted to the top 1 mm of the superficial layer of articular cartilage as previously described²⁴.

Important issues to address in future studies relate to the long-term effects of the ultraviolet-A treatment and how long target gene expression persists. We did not identify any evidence of articular chondrocyte apoptosis after one week. Considering that this programmed cell death is an immediate response to environmental stress²⁵, it is unlikely that ultraviolet A could induce apoptosis after seven days. However, it is possible that this treatment could induce delayed radiation fibrosis, which will need to be investigated. In terms of the length of gene expression, we and other investigators have found that rAAV transduction of articular chondrocytes is transient^12,24. Additionally, rAAV is replication-defective and rapidly diluted from cell division in healing tissue²⁶. Thus, our expectation is that clinically important gene expression will last less than three weeks.

A surgical procedure that enables adult articular cartilage to reactivate the developmental process and heal, such as is observed in other tissues following damage, has been elusive. It is our belief that our findings give promise for a future advance that would provide a means for the regeneration of articular cartilage to an injured or degenerative joint. ?

References

Materials and Methods | Results | Discussion | References

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Topics

gene therapy ; cytotoxic drug therapy ; fibroblast ; articular cartilage ; chondrocytes ; dna ; pyrimidine dimers ; reactive oxygen species ; safety ; synoviocytes

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Michael D. Maloney, J. Jeffrey Goater, Richard Parsons, Hiromu Ito, Regis J. O'Keefe, Paul T. Rubery, M. Hicham Drissi, Edward M. Schwarz; Safety and Efficacy of Ultraviolet-A Light-Activated Gene Transduction for Gene Therapy of Articular Cartilage Defects. The Journal of Bone & Joint Surgery. 2006 Apr;88(4):753-761.

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