Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II
© Tang et al.; licensee BioMed Central Ltd. 2010
Received: 16 December 2009
Accepted: 8 July 2010
Published: 8 July 2010
Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA).
We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence.
We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint.
Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.
Rheumatoid arthritis (RA) is a systemic disease with polyarticular manifestation of chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. The current systemic anti-TNF-α therapies ameliorate disease in 60% to 70% of RA patients . However, biologics must be given systemically in relatively high dosages to achieve constant therapeutic levels in the joints, and significant side effects have been reported .
Gene therapy may provide an effective alternative to drug delivery for the treatment of arthritis . Although various strategies have been tested, those that target gene delivery to the synovial lining of joints have made the most experimental progress [3, 4]. This strategy has shown efficacy in several experimental models of RA [5–7]. For this reason, we have developed a unique modification to a clinically acceptable method of gene delivery to allow delivery of the gene product directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) of type II collagen (CII) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to CII and arthritis in the collagen-induced arthritis (CIA) model . Such collagen peptides containing specially designed substitutions and expressed as a gene products may provide an ideal choice to be delivered to the joints.
We engineered an adenoviral gene-based therapy and showed that this treatment strategy provided a sustained, local therapy for individual arthritic joints. Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells . They are potentially safer than current therapies because they contain a modification of an endogenous naturally occurring protein, used to interrupt the autoimmune T cell attack and allow for tissue repair. We believe this approach has the potential to become applicable for treatment of RA.
Materials and methods
Preparation of tissue-derived type II collagen
Native CII was solubilized from fetal calf articular cartilage by limited pepsin-digestion and purified as described earlier . The purified collagen was dissolved in cold 0.01 M acetic acid at 4 mg/ml and stored frozen at -70°C until used.
DBA/1 mice were obtained from the Jackson Laboratories and raised in our animal facility. They were fed standard rodent chow (Ralston Purina Co., St. Louis, MO, USA) and water ad libitum. The environment was specific pathogen-free and sentinel mice were tested routinely for mouse hepatitis and Sendai viruses. All animals were kept until the age of 7 to 10 weeks before being used for experiments, which were conducted in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols.
CII was solubilized in 0.01 M acetic acid at a concentration of 4 mg/ml and emulsified with an equal volume of complete Freund's adjuvant (CFA) containing 4 mg/ml of Mycobacterium tuberculosis strain H37Ra (Difco Microbiology Products, Becton Dickinson, NJ, USA) . Each mouse received 100 μg of CII emulsified in CFA intradermally at the base of the tail.
Generation of replication-defective, recombinant adenoviral vector expressing modified CB11
Recombinant adenovirus carrying cDNA for rCB11-A9 was generated using a BD Adeno-X Expression System (BD Biosciences Clontech (San Jose, California, USA)), which incorporates the rCB11-A9 expression cassette into a replication-incompetent (ΔE1/ΔE3) human adenoviral type 5 (Ad5) genome. All work was conducted in accordance with approved Institutional Biosafety Committee (IBC) protocols. In brief, an 834 bp of full-length murine CB11 gene was PCR-amplified from murine spleen cDNA and cloned into the PCR2 vector (Invitrogen, Carlsbad, California, USA). We introduced three point mutations (I260A, A261B, and F263N) within the immunodominant T cell determinant of CB11 (CII124-402) to generate an rCB11-A9 construct. The rCB11-A9 cDNA was then excised with BamHI/EcoR I and subcloned into the same sites of the pShuttle2 vector to construct an rCB11-A9 specific expression cassette. For in vivo bioimaging analysis, a cDNA encoding the luciferase gene was also subcloned into the pShuttle2 to establish the Adeno-X-luciferase expression cassette. To produce recombinant adenoviral DNA containing rCB11-A9 or luciferase, we excised the expression cassettes from recombinant pShuttle2 plasmid DNA by digesting with I-Ceu I and PI-Sce I and ligated the expression cassettes with prelinearized BD Adeno-X Viral DNA (I-Ceu I and PI-Sce I digested). Low passage HEK293 cells were transfected with the resultant recombinant adenoviral DNA using the calcium phosphate method . The recombinant adenoviral particles were harvested by lysing transfected cells. The resultant AdenoX-rCB11-A9 is a replication-incompetent recombinant adenovirus. High titer viral stocks (about 108 to 109 plaque forming units (pfu)/ml) were obtained by amplifying recombinant adenovirus in HEK 293 cells. A construct (pShuttle2-lacZ) was included in the BD Adeno-X Expression System and recombinant AdenoX-lacZ was generated as described above and used as a control. The recombinant adenoviral titers were determined by BD Adeno-X Rapid Titer Kit [11, 12].
Production and purification of recombinant CB11 and CB11-A9
In some experiments, a baculoviral expression system was used to produce rCB11 (CII124-402bac) in insect cells essentially as described earlier . The cDNA for both recombinant CB11 and CB11-A9 (rCB11 and rCB11-A9) were subcloned into a Gateway entry vector (Invitrogen, Carlsbad, California, USA) and validated. The resultant Gateway entry vectors containing either rCB11 or rCB11-A9 were ligated with BaculoDirect Linear DNA (Invitrogen, Carlsbad, California, USA) and transfected into Sf9 insect cells. Supernatants from lysed insect cells were collected and screened for expression by performing SDS-PAGE and western blot analysis. After validated, high titers of recombinant baculovirus were obtained by re-infecting Sf9 cells twice and supernatants collected from lysed cells. To express the recombinant proteins Hi5 cells was infected with high titer of baculovirus. Supernatants from cultured Hi5 cells were harvested by centrifugation and the recombinant proteins purified by gel filtration and cation exchange chromatography, and dialyzed in dilute acetic acid.
The hind ankle joints of DBA/1 mice were injected intra-articularly with 10 ul of adenoviral vector 1 × 107 pfu of adenovirus, containing the DNA for either luciferase, rCB11-A9, or Lac-Z. In some experiments, selected mice were injected intraperitoneally with luciferin, and the expression of the transgene (luciferase) was detected by bioluminescent imaging using a liquid nitrogen cooled CCD camera (Photometric Chemipro, Roper Scientific, NJ, USA) mounted on a dark box one hour later. Images were acquired and analyzed using Metamorph software (Universal Imaging Co., Dowlington, PA, USA).
Measurement of the incidence and severity of arthritis
The incidence and severity of arthritis were determined by visually examining each forepaw and hindpaw and scoring them on a scale of 0 to 4 as described previously . Scoring was conducted by two examiners, one of whom was unaware of the identity of the treatment groups. Each mouse was scored thrice weekly beginning three weeks post immunization and continuing for eight weeks. The incidence of arthritis (number of animals with one or more arthritic limbs) and mean severity score (sum of the severity scores/total number of animals in the group) was recorded at each time point.
In a prevention protocol, four groups of 10 DBA/1 mice each were administered intra-articularly in the ankles, either adenoX-rCB11-A9 or adenoX-LacZ. The mice were immunized with CII/CFA either three or seven days after the injection.
In a treatment protocol, groups of three DBA/1 mice were immunized with CII/CFA and at the time arthritis reached a severity score of two or greater, the mice were administered intra-articularly in the hind ankles either adenoX-rCB11-A9 or adenoX-LacZ.
Measurement of serum antibody titers
Mice were bled at six weeks after immunization and sera were analyzed for antibodies reactive with native CII using a modification of an ELISA previously described . Serial dilutions of a standard serum were added to each plate. From these values, a standard curve was derived by computer analysis using a four-parameter logistic curve. Results are reported as units of activity, derived by comparison of test sera with the curve derived from the standard serum which was arbitrarily defined as having 50 units of activity. Reactivity to CII was not detected in sera obtained from normal mice.
Measurement of cytokines
Groups of three DBA/1 mice were administered intra-articularly either adenoX-rCB11-A9 or adenoX-LacZ and the mice were immunized with CII/CFA three days after the injection. Draining lymph node cells were harvested 14 days after the immunization and cultured (5 × 106 cells/ml) with 100 μg/ml of either the mouse collagen immunodominant peptide, Ova (negative control), or purified protein derivative (PPD) (positive control). Supernatants were collected 72 hours later and analyzed for the presence of multiple cytokines (IL-4, IL-5, IL-10, IL-2, interferon (IFN)γ, and IL-17 by a Bio-plex mouse cytokine assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Values are expressed as picograms per ml and represent the mean values for each group.
The incidence of arthritis in various groups of mice was compared using Fisher's Exact Test. Mean severity scores and antibody levels were compared using Student's t test.
An adenoviral construct efficiently transfers an exogene into arthritic synovial tissues in collagen-induced arthritis
Development of the baculovirus construct for modified collagen (rCB11-A9) expression and evaluation of its immunogenicity
A9-modified recombinant CB11 is not arthritogenic
Format of collagenous immunogena
Antibodies to CIIc
2.5 ± 1, P < 0.05
21.3 ± 5
Development of a method for in vivobioimaging to track the duration of gene expression
Evaluation of the potency of the AdenoX-rCB11-A9 construct in suppression of CIA
AdenoX-rCB11-A9 treatment suppresses anti-CII antibodies
Antibodies to CII in treated mice
Antibodies to CIIb
AdenoX-rCB11-A9 (imm CII/CFA 3 days later)
19.6 ± 2, P < 0.05
AdenoX-rCB11-A9 (imm CII/CFA 7 days later)
16.2 ± 2 P < 0.005
AdenoX-lacZ (imm CII/CFA 3 days later)
37.2 ± 10
AdenoX-lacZ (imm CII/CFA 7 days later)
45.0 ± 9
Mechanism of suppression
Cytokine responses in mice treated with gene therapy
131 ± 12
137 ± 14
280 ± 22
1 ± 1
82 ± 10
18 ± 8
1,405 ± 120
643 ± 25
6,364 ± 220
7 ± 3
762 ± 44
214 ± 17
631 ± 50
2,451 ± 50
8,612 ± 267
3 ± 2
820 ± 63
262 ± 21
120 ± 15
164 ± 22
250 ± 25
2 ± 1
75 ± 8
16 ± 9
152 ± 14
143 ± 15
286 ± 25
44 ± 6
203 ± 21
47 ± 11
776 ± 62
2,620 ± 232
8,633 ± 547
2 ± 2
778 ± 83
245 ± 26
Potency of AdenoX-rCB11-A9 in suppression of CIA when injected after the onset of arthritis
Our aim was to engineer an adenoviral-based therapy designed to make synovial cells secrete a modified naturally produced molecule, type II collagen, thereby providing a sustained, local therapy for individual arthritic joints. This approach is attractive because joints are discrete, accessible cavities that can be readily injected. Many different genes have been evaluated for their ability to treat animal models of RA . These have led to several clinical trials, confirming the feasibility and in a preliminary fashion, safety of gene transfer to human arthritic joints [3, 4, 18, 19].
Recently, several studies using adenoviral-mediated gene transfer of therapeutic genes for animal model treatment have been reported [7, 20–22]. Adenoviruses carry their genetic material in the form of double-stranded DNA. When these viruses infect a host cell, the DNA molecule is left free in the nucleus of the host cell, and is transcribed, but not replicated. The advantages of this therapy are two-fold . The treatment gives a sustained release of the material directly into the joint cavity, greatly decreasing the amount of material required and the number of injections necessary. Second, the absence of integration into the host cell's genome lessens the possibility of permanent side effects, and prevents the possibility of malignant-type transformations. Although concerns about the safety of adenovirus vectors have been raised, newer genetically crippled versions of the virus together with modified or deleted capsid sequences have demonstrated an increased safety and potential for stable transgene expression .
Most gene transfer strategies for treatment of RA are currently broad based, designed for introducing cytokines [3, 4, 18–22]. The capability of collagen peptides to act locally to induce T cells to secrete suppressive cytokines in a limited environment makes them interesting as potential therapeutic reagents in suppressing RA. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that profoundly suppressed immunity to CII and arthritis. In a mouse model of RA, A9 protein therapy achieved a dramatic arrest in the overall disease progression as judged by clinical, histopathological, and immunological manifestations of arthritis . We now demonstrate in vivo immunomodulatory properties of rCB11-A9, supporting its therapeutic potential in the treatment of inflammatory autoimmune disorders. Such collagen peptides containing specially designed substitutions and expressed as gene products may provide an ideal choice to be delivered to the joints. The advantages over conventional therapies include the ease with which they can be injected at the site of the inflammation, targeting the specific arthritogenic lymphocytes that initiate and perpetuate joint inflammation, and transforming potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells. Our results suggest that the effects are primarily localized to the joints, although we have not performed biodistribution studies. They are potentially safer than current therapies because they contain a modification of an endogenous naturally occurring protein. The use of the gene therapy overcomes the problems of rapid degradation and short half-life of small synthetic proteins in vivo.
Another great advantage of gene delivery to the synovial cells is that they contain the enzymatic apparatus to apply post-translational modifications, including the hydroxylation and glycosylation of lysine residues, which occur in chondrocyte synthesized CII, but not synthetic peptides. It is known that CII peptide fragments derived from the cyanogen bromide digestion of native CII are immunologically more active than chemically synthesized peptides [24, 25]. It is now generally accepted that part of the T cell response to cartilage-derived CII is dependent upon the presence of glycosylated determinants, which stabilize major histocompatibility complex/T cell receptor (MHC/TCR) interaction or act as part of the epitope [24–27].
Despite these advantages, it should be noted that there is no consensus concerning the ideal vector for human gene therapies. For example, patients can carry pre-existing neutralizing antibodies to adenoviral vectors or develop them after the first injections, reducing their effectiveness. Although scientific breakthroughs continue to move gene therapy toward mainstream medicine, future research should enhance clinical applications of a collagen-based gene therapy for RA.
In summary, our studies demonstrate that: recombinant CB11-A9 adenovirus can efficiently transfer and express exogenes in joints and synovial tissue; the expression persists for at least 18 days after the injection; and this type of therapy is effective at both prevention and treatment of autoimmune arthritis. These data strongly support our hypothesis that adenoviral-mediated modified collagen-type therapies can suppress arthritis and transform activated T cells and bystander T cells into therapeutic (regulatory-like) T cells. Gene therapy has emerged as an effective and promising therapeutic strategy for RA . To this end, local gene delivery can provide an alternative approach to achieve high, long-term expression of biologics, optimizing the therapeutic efficacy and minimizing systemic exposure. Future analogs can be optimized for binding to the human MHC . Our data using adenoX-rCB11-A9 in the CIA animal model convincingly supports the possibility of a collagen-based gene therapy for RA.
type II collagen
complete Freud's adjuvant
enzyme-linked immunosorbent assay
Institutional Animal Care and Use Committee
Institutional Biosafety Committee
major histocompatibility complex/T cell receptor
polymerase chain reaction
plaque forming units
purified protein derivative
tumor necrosis factor.
This work was supported, in part, by USPHS Grants AR-55661, AR-55266, and program-directed funds from the Department of Veterans Affairs and the Arthritis Foundation.
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