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This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract This study examined the influence of osteoprotegerin OPG gene transfer on a murine collagen-induced arthritis model.
A single periarticular injection of AAV-OPG or AAV-LacZ on the arthritic paw successfully incorporated the exogenous gene to the local tissue and resulted in marked transgene expression in the joint homogenate for at least three weeks.
Clinical disease scores were significantly improved in OPG treated mice starting at day post-treatment. Histological assessment demonstrated that OPG gene transfer dramatically protected mice from erosive joint changes compared with LacZ controlsalthough treatment appeared less effective on the local inflammatory progress.
Overall, gene transfer of OPG effectively inhibited the arthritis-associated periarticular bone erosion and preserved the architecture of arthritic joints, and the study provides evidence that the cartilage protection of the OPG gene therapy may be associated with the down-regulation of MMP3 expression.
Introduction Rheumatoid arthritis RA is one of the most common forms of arthritis that affects more than two million Americans. It is characterized by chronic inflammation of the joint s with severe pain and swelling, impaired functions, and eventually joint distortion [ 1 — 3 ].
Bone destruction is a frequent and clinically serious event in patients with RA.
RANKL expression was documented at the sites of active bone erosions [ 4 ]. RANKL functions both as a membrane anchored molecule and a soluble molecule. RANK is expressed on osteoclast precursors and mature osteoclasts [ 15 ].
RANKL also serves as survival factor and activates osteoclasts, suggesting its important role in the progression of rheumatoid arthritis.
OPG was initially identified as a soluble factor that was capable of inhibiting osteoclastogenesis in vitro [ 1920 ]. OPG-deficient mice exhibit severe osteoporosis [ 21 ].
Due to the short half-life of biological agents and the chronic nature of the disease, gene therapy provides an attractive alternative to deliver gene to arthritic site where therapeutic protein OPG could be locally produced and executed.
Materials and Methods 2. All animals were quarantined in the facility for two weeks prior to experimentation. Onset of CIA was determined upon observation of the first signs of definitive erythema and edema in the metatarsal or metacarpal regions of the paws. Arthritic animals were clinically assessed 5 times a week and paw measurements were recorded 3 times a week for 8 weeks.
The severity of arthritis in the affected paw was graded according to an established score system [ 2526 ] as follows: The maximum score per mouse is 12 4 paws representing overall disease severity and progression in the animal.
All animals were sacrificed at week 8 after disease onset except the group of mice for detection of transgene expression described in next paragraph. The arthritic paws were harvested and divided into 2 parts along the median longitudinal axis.
Tests were performed using the standardized protocol previously described [ 2728 ], and the protein levels were estimated against the standard curve corun on the same ELISA plate. Toluidine blue staining was also performed for cartilage damage evaluation.
The threshold was kept constant for all samples to eliminate the noise from the soft tissue. Joints were harvested from arthritic mice postmortem at 56 days after arthritic onset.
The cDNA was reverse transcribed from 0. The primer pairs of the target genes used in this study Table 1 were designed using a software Primer3 http: The values of threshold cycle Ct at which a statistically significant increase in reporter-dye signals is first detected, were recorded to calculate the relative quantification of target gene expression.
The reaction mixture after real-time PCR was electrophoresed on 1. Primer sequences for the genes used in this study.
A -value of 0. In virus-transduced arthritic joint homogenate, human OPG levels were maintained for at least 21 days, with the peak at 7 days following gene transfer Figure 1.
OPG protein level in blood serum was minimum, showing local effects of therapy. Mouse paws receiving AAV-lacZ vector injections revealed positive blue colored LacZ expressing cells under X-gal stains, similar to the reported previously [ 25 ].
There was no evidence of X-gal positive staining in uninjected paws or remote organs. ELISA was performed to determine the transgene expression. Effects of OPG Gene Transfer on Clinical Progression of CIA Therapeutic gene transfer of OPG significantly ameliorated the mean numbers of arthritic paws of each mouse during the disease progression Figure 2 a and the mean arthritis scores of arthritic paws since the day that the first paw had arthritic changes Figure 2 b.
The OPG gene transfer significantly diminished the severity of the disease progress as early as 3 weeks after onset. Panel a shows the mean numbers of arthritic paws of each mouse during the disease progression, while panel b illustrates the arthritis scores of arthritic paws from the day of disease onset.
Histological Assessment of CIA following OPG Gene Modification Histological evaluation of the arthritic paws of negative control or LacZ-transduced mice revealed severe synovial inflammatory cellular infiltration and pannus formation, marked bone and cartilage erosion, and distortion of joint architecture Figure 3.When these cells behave in an abnormal manner, it can lead to acute myeloid leukemia.
Various gene therapy-based tests have been conducted on the guinea pigs as model animals. The gene therapy-based experiments have shown that deafness can be cured with the help of gene therapy.
Results have indicated various target organs and gene transfer techniques resulting to a wide scientific and medical acceptance regarding the feasibility of gene therapy for disorder of various organs such as liver, lungs and some kind of cancer in addition to enzymes and hormones.
Gene therapy poses many technical questions. These include regulation of the activity of the transferred gene to produce the appropriate amount of the gene product at the right time and place.
In addition, the insertion of the therapeutic gene must not harm other necessary cell functions. Sample Assessment Materials For international centres only.
Further testing of gene therapy is under way, and the tests are run in the same way as for any drug. These tests involve several stages or phases. a major reaction, but as the scientists raise the dose, the potential for problems increases. The results show that the majority of respondents are supportive of the process of gene therapy for disease prevention and health purposes, but most are very against the use of .
In spite of benefits, gene therapy has some risks for patients and society. The information provided by genetic testing can have profound effects on the lives of those involved, for example in the areas of health insurance and employment.
In some cases, germline gene therapy could violate human rights and .