Gene therapy constitutes a promising strategy for the treatment of osteoarthritis (OA). We assessed the use of electroporation (EP) of non-viral gene vectors, and compared its efficacy with that of adeno-associated virus (AAV) vectors. EP- and AAV-mediated delivery of human interleukin-1 receptor antagonist (hIL-1R a) was localized performed in the joints of rats following induction of OA. mRNA levels for hIL-1Ra, IL-1 β, TNF-α, MMP-13 and ADAMTS-4 in the cartilage and synovial tissues were analyzed. Structural analyses of the subchondral bone at the medial femoral condyle were performed by Micro-CT after treatment. Knee joint specimens were staining with hematoxylin and eosin and Saffron O. Induction of hIL-1Ra by both EP and AAV inhibited inflammatory-induced sub-chondral bone reconstruction, and effectively suppressed IL-1β activity, as evidenced by decreased expression of MMP-13 and ADAMTS-4. Histological analyses revealed significant protection of cartilage, proteoglycan by EP and AAV. hIL-1Ra expression was similar in both the EP and AAV groups. Notably, this gene is not easier degraded transduced by EP compared with AAV. Taken together, these results show that EP offers transfection efficiency comparable to that of AAV, with the potential for longer gene expression, making EP a promising candidate for efficient non-viral delivery of OA gene therapy.
Osteoarthritis (OA) is the one of the most common joint diseases, characterized by loss of articular cartilage in combination with underlying bone changes, including sclerosis, bone cysts, subchondral bone sclerosis, and osteophyte formation [
As a biological alternative for arthritis-based therapies, many studies have used gene vectors to treat arthritis in various animal models [
Designing an efficient protocol for OA treatment using non-viral gene delivery demands a number of considerations, including both the gene of interest for OA therapy, and the method for gene-delivery, with a need to balance both safety and efficacy. Interleukin-1 (IL-1) is the major cytokine involved in the pathology of OA [
Recently, electroporation (EP) has been identified as a safe and effective non-viral gene delivery method both in vitro and in vivo [
Animal studies were performed using 8-week old Male Sprague Dawley (SD) rats, weight 220 - 250 g, which were provided by the Experimental Animal Center of Chinese People’s Liberation Army (PLA) General Hospital. All protocols were approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital. The animals were randomized into treatment protocol groups. OA was induced by surgery of medial meniscus resection and anterior cruciate ligament transection, 1 week later, experimental OA pathology gradually developed over the surgery. Electroporation and AAV transfection of hIL-1Ra was initiated 1 week after the operation. Following treatment, knee joints were isolated once a week for the course of the study, and analyzed using histological, morphological, radiological, and molecular analyses. It is worth noting, in Results part, the 1-week time point represent 1 week after gene transfer not means 1 week after OA induced surgery.
Rats were randomly divided into 5 groups. Group 1: untreated, non-arthritic rats (normal group, n = 24). Group 2: untreated arthritic rats that underwent OA surgery, with no follow up treatment (OA group, n = 24). Group 3: OA rats treated with the hIL-1Ra/pCI-neo plasmid without transfection (naked plasmid (NP) group, n = 24). Group 4: OA rats electroporated with the hIL-1Ra/pCI-neo plasmid (electroporation (EP) group, n = 24). Group 5: OA rats treated with an adeno-associated virus (AAV) carrying the hIL-1Ra/pCI-neo plasmid (AAV group, n = 24). For groups 3 and 4, the dose of hIL-1Ra plasmid was acquired by Result 3.1.
Luciferase (Luc) was used as a reporter gene for bioluminescence observation. A luciferase plasmid was purchased from Genomeditech Co. (GM-0210PC) and injected into both sides of the rat knee joint through the patellar tendon. In vivo electroporation of the right joint was then used for transfection of this gene into cells.
To obtain bioluminescence images, rats were anesthetized using a nose cone delivering 2% isoflurane in 100% O2. D-Luciferin (40902ES03) was diluted to 30 mg/mL in Dulbecco’s phosphate buffered saline (60147ES76), and 100 µL D-Luciferin was directly administrated into both joints 1, 3, 7, 14, 21, and 28 days post-electropo- ration. Next, the rats were placed in a light-obstructing chamber for 10 min, and luciferase kinetics was recorded by consecutive 5 min acquisitions. A number of highlighted pixels were then counted using the instrument’s imaging unit software to quantify the bioluminescent signal in the area shaped around each site of photon emission.
Human IL-1Ra cDNA was generously provided by Prof. Yu CL (Institution of Sports Medicine, Peking University Third Hospital, China). Full-length human IL-1Ra was cut from pcDNA3.1 (Invitrogen, USA). The human IL-1Ra plasmid was used in the treatment of OA, as described previously.
Rats in groups 2 - 5 were anesthetized using a nose cone delivering 2% isoflurane in 100% O2. For groups 3, 4, a mixture of hIL-1Ra plasmid in 100 µL phosphate-buffered saline (PBS) was injected into the right knee joint using a syringe equipped with a 30-gauge needle; group 2 was treated with 100 µL PBS. Following injection, the knee joint was flexed and extended several times. A pair of 8 mm electrode pads was then placed on the joint in group 4, as show in
For group 5, human IL-1Ra was also packaged into AAV vectors, the serotype is type 2, which were purified and titered as described previously. After packeaged, the delivery of AAV vectors, 2 × 109 vector genomes were diluted into 100 µL solution, were directly injected into articular cavity, to achieve transfection in vivo. Not using AAV vectors transfect special cells, such as synoviocytes or chondrocytes or stem cells, in vitro, and then inject those cells into articular cavity.
Following isolation of the knee joints, the articular cartilage and the synovium were separated, as described previously. Isolated tissues were then immersed in liquid nitrogen immediately in order to minimize RNA degradation. Total RNA was extracted in 1 mL TRIzol solution (Invitrogen, USA) for 24 h at 4˚C. cDNA was generated using the ReverTra Ace qPCR RT Kit (Toyobo, Japan) at 37˚C for 15 min, and at 98˚C for 5 min. Real-time PCR was performed using the SYBR Green Realtime PCR Master Mix-Plus (Toyobo). For each reaction, 1 µL cDNA and 1 µL of each primer were used. The primer sequences used are as follows: rat GAPDH forward primer 5’-ATGGTGAAGGTCGGTGTGAACG-3’ and reverse primer 5’-TTACTCCTTGGAGGCC ATGTAG-3’; hIL-1Ra forward primer 5’-AGATGTGCCTGTCCTGTGTC-3’ and reverse primer 5’-CCTGC TTTCTGTTCTCGCTC-3’; rat IL-1β forward primer 5’-TTGTGGCTGTGGAGAAGCTG-3’ and reverse primer 5’-GCCGTCTTTCATACACAGG-3’; rat TNF-α forward primer 5’-CATGATCCGAGATGTGGAACT
GGC-3’ and reverse primer 5’-CTGGCTCAGCCACTCCAGC-3’; rat ADAMTS-4 forward primer 5’-AGA GTCCGAACGAGTTTACG-3’ and reverse primer 5’-GTGCCAGTTCTGTGCGTC-3’; rat MMP-13 forward primer 5’-CCCAGCCCTATCCCTTGATGCCA-3’ and reverse primer 5’-TGCAGGCGCCAGAAGAATCTG T-3’. PCR amplification reactions were run in a total volume of 20 μL. The reaction conditions were as follows: enzyme activation at 95˚C for 1 min, followed by 40 cycles of denaturation at 95˚C for 15 s, and all the primers annealing temperatures are 57˚C for 15 s and extension at 72˚C for 45 s. Results were recorded and analyzed using StepOne software v2.1 (Applied Biosystems, USA). Expression was normalized relative to GAPDH. Each experimental group consisted of three knee joints, and was repeated three times for each time point.
A resolution microtomograph scanner was used to assess both qualitative and quantitative changes to the subchondral bone at each time point following induction of OA. First, rat knee joints fixed in 4% neutral paraformaldehyde were loaded into a Micro-CT holder. X-ray parameters were then manually adjusted to values at which mineralized tissue could be visualized. The joints were scanned a 360˚ rotation at a 45 µm resolution. A MicroView 3D image viewer (GE Healthcare, USA) was used to reconstruct the scan data and visualize the trabecular and subchondral bone. A cylindrical region of interest (ROI) was selected from the weight-bearing area of the medial femoral condyles for analysis. The volume and diameter of the trabecular bone were measured as trabecular thickness (Tb.Th), mean trabecular separation (Tb.Sp), bone volume per tissue volume (BV/TV), and bone mineral density (BMD).
The Catwalk equipment consisted of an enclosed walkway with a glass platform that was illuminated by a completely internal reflection of green light. Whenever the rat’s paw made contacted with the glass platform, green light was able to escape, and the area of illumination could be captured using Illuminated Footprint technology consisting of a high speed video camera positioned underneath the walkway. The home cage was located at the end of the walkway. Rats were placed in the entrance of the channel and allowed to walk freely across the glass into the home cage. Results were recorded and analyzed using the Catwalk XT 8.1 Software. The goal of this experimental approach was to measure 3D footprint intensity, which consists of all four paws within an individual frame. Intensities ranged from 0 to 225 and were identified by different colors. Differences in intensity are considered representative of movement-evoked pain in OA.
Isolated knee joints were prepared for histological examination and immunohistochemistry (IHC). First, each joint sample was photographed for evaluation of cartilage repair. After gross examination, tissues were fixed in 4% neutral paraformaldehyde for 7 days, and decalcified in 10% neutral ethylene diamine tetraacetic acid (EDTA) for 4 weeks at 37˚C. Then, the samples were dehydrated using an alcohol gradient and embedded in paraffin blocks. Finally, samples were cut into 7 mm sections using a microtome (Leica Microsystems, Germany). Representative sections were stained with hematoxylin and eosin (H-E) and Safranin orange for histological analysis.
Rat articular cartilage sections were incubated with rabbit monoclonal anti-hIL1Ra antibody (Abcam, ab124962) or rabbit anti-COL II antibody (Abcam, ab34712) overnight at 4˚C, and then incubated with goat anti-rabbit secondary antibodies (MaxVison, 150304407E). Stained samples were photographed digitally and observed under confocal microscopy (Olympus, Japan).
All quantitative date are expressed as mean ± SD. A Tukey test was used to assess differences between groups. P values ≤ 0.05 were considered statistically significant.
Few previous studies have described transgene expression after plasmid transfer to the joint by EP [
fection efficiency of EP. Bioluminiesence signals were clearly observed in all groups, with representative images of group C (50 µg, 30 ms) shown in
The mean intensity (weight load) of the right hind paw while walking decreased significantly over the total test period in the both OA and NP groups (naked plasmid (NP) group) relative to the normal group (
To examine transfection efficiency and explore interconnectedness of OA associated inflammatory factors, we determined the expression level of hIL-1Ra, IL-1β, TNF-α, MMP-13, and ADAMTS-4 in extracts from rat knee joint using PCR analysis. mRNA expression of IL-1Ra was significantly up-regulated in both the EP and AAV groups relative to other groups at each time point (
Group | 1W | 2W | 3W | 4W |
---|---|---|---|---|
OA | 103.94 ± 1.45* | 99.04 ± 3.62* | 78.24 ± 1.29* | 66.56 ± 5.66* |
NP | 113.79 ± 5.60*# | 93.15 ± 3.34* | 76.89 ± 1.19* | 68.72 ± 2.07* |
EP | 124.30 ± 2.26#△ | 120.80 ± 2.01*#△ | 118.79 ± 0.60*#△ | 119.25 ± 3.28*#△ |
AAV | 124.88 ± 3.02#△ | 128.35 ± 4.41#△ | 117.57 ± 4.65*#△ | 110.50 ± 2.71*#△ |
Control | 127.58 ± 1.61 | 130.00 ± 2.05 | 136.70 ± 6.97 | 139.66 ± 5.34 |
Values are mean ± SD. Repeated measures ANOVA, with Tukey test used compared with normal rats, *p < 0.05; compared with OA group rats, #p < 0.05; compared with NP group rats, △p < 0.05, at each time point.
than EP, but is more susceptible to degradation and inactivation. mRNA expression was significantly higher in the NP group relative to both the OA and normal groups at 1 week; no statistically significant differences were observed at any subsequent time points, suggesting that the naked plasmid was not the source of any desired therapeutic effect.
Consistent with its role as the primary factor in the OA inflammatory cascade, IL-1β expression gradually increased in OA rats throughout the course of this experiment. In both the EP and AAV groups, IL-1β mRNA expression was higher than that of the normal group, though significant differences did not appear until week 4. In the NP group, IL-1β mRNA levels were significantly different compared to the OA group at 1 week. Naked IL-1Ra plasmid was able to confer modest suppression of inflammation at week 1, but the efficiency of this treatment was quickly lost (
TNF-α mRNA levels were strongly correlated with an increase in OA-mediated joint swelling [
MMP-13 and ADAMTS-4 are important downstream targets of both IL-1β and TNF-α [
To better understand the pathology of trauma-induced OA in rats, we used Micro-CT to analyze changes in the subchondral bone. In sites of severe arthritis, the subchondral bone becomes thicker [
to each of these parameters, no significant differences were seen between the OA and NP groups, though all were significantly higher than the normal group, the only exception being the BV/TV score in the NP group, which was not significantly different from the normal group at 1 week. In contrast, each of these values were significantly lower in both the EP and AAV groups, relative to the OA and NP groups over the entire test period. While all three of these values did increase over time, the rate of increase was significantly lower than that of either the OA or NP groups (
Gross evidence of cartilage damage was assessed in each of the five groups at 4 weeks after treatments (
To evaluate the cartilage damage, histological staining was done 1, 2, 3 and 4 weeks after treatment (
treatment, crack depth in the subchondral bone and chondrocytes had become extremely disordered, with the cartilage almost completely destroyed, and the subchondral bone plate shifted (
In the
indicating a possible up-regulation of IL-1Ra at this time point. As the major collagen present in the chondrocyte extracellular matrix, type II collagen was gradually degraded during OA progression (
OA progression is a complex process that includes multiple cellular and molecular events that take place in a specific spatial and temporal order. Regulation of inflammatory processes in arthritis is driven in part by IL-1 and its natural inhibitor IL-1Ra [
The data presented here show that IL-1Ra can be successfully transduced in vivo, overcoming important technical challenges in terms of safety, efficacy, and convenience for transgene delivery [
Initial validation of EP for intra-articular delivery required optimization of delivery parameters for both pulse width and plasmid dose. In vivo bioluminescence imaging was used to detect the fluorescence following transfection with a plasmid encoding luciferase [
AAV-mediated delivery is a well-established model for gene therapy, having been studied in numerous diseases [
A careful analysis of effects of OA inhibition is indispensable for assessing the success of EP as a transfection technique. In this study, both μCT and histological analyses revealed progressive destruction of the medial femoral condyle cartilage and subchondral bone induced by OA. Catwalk gait analysis was used to correlate joint damage with movement-evoked pain in OA.
BMD, BV/TV, Tb.Th scores obtained for the subchondral bone underlying the weight-bearing areas of the medial femoral condyle were significantly lower in the EP and AAV groups relative to untreated OA controls. Taken together, these data demonstrate similar efficacy for both EP and AAV-mediated delivery of IL-1Ra in terms of delay the remodeling of subchondral bone. While the timing and relationship between cartilage destruction and subchondral bone reconstruction remains poorly understood [
Little difference is seen in terms of µCT analysis of the subchondral bone or expression of inflammatory markers of transfected mice, including IL-1β, TNF-α, MMP-13, and ADAMTS-4 over the first three weeks. While none of these factors were expressed at levels significantly higher than that of normal controls, both BMD and Tb.Th were, implying that the subchondral bone reconstruction may be occurring prior to inflammation of the cartilage and synovium.
After electroporation of IL-1Ra into the articular, inflammatory related factors IL-1β, TNF-α, MMP-13, and ADAMTS-4 were all strongly downregulated. IL-1β has been shown to induce chondrocytes to produce metalloproteinases, mainly interstitial collagenase 3 (MMP-13), a family of enzymes that play an important role in the degradation of cartilage components [
Continued expression of target gene IL-1Ra was seen in both the AAV and EP groups; however, a sharp decrease was seen in the AAV group compared with that of the EP at four weeks post-treatment [
The use of EP as an efficient method for therapeutic gene delivery into the intro-articular tissue, with gene expression levels similar to that of AAV, with expression for longer periods of time. Given the well-documented safety issues associated with AAV delivery, EP may constitute an effective alternative for gene delivery in OA.
Funding: This work was funded by Ministry of science and technology project 973(2014CB542201); Beijing municipal science and technology projects (Z141107004414044); National high technology research and development program of 863 (2012AA020502); National Natural Science Foundation of China (81572148).
Financial interest: There is no conflict of interest in this section.
The authors gratefully acknowledge the assistance of Feng Xu of the department of Orthopedics, General Hospital of People’s Liberation Army, Beijing, People’s Republic of China, for their contribution in surgery procedures.
Zhen Sun,Heyong Yin,Xiaoming Yu,Xun Sun,Bo Xiao,Yichi Xu,Zhiguo Yuan,Haoye Meng,Jiang Peng,Changlong Yu,Yu Wang,Quanyi Guo,Aiyuan Wang,Shibi Lu, (2016) Inhibition of Osteoarthritis in Rats by Electroporation with Interleukin-1 Receptor Antagonist. Journal of Biomedical Science and Engineering,09,323-336. doi: 10.4236/jbise.2016.97027