Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors

Sirkka Kyostio-Moore Sanofi, 49 New York Ave, Framingham, MA 01701.

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Patricia Berthelette Sanofi, 49 New York Ave, Framingham, MA 01701.

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Cathleen Sookdeo Cornell Sanofi, 49 New York Ave, Framingham, MA 01701.

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Bindu Nambiar Sanofi, 49 New York Ave, Framingham, MA 01701.

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Monica Dias Figueiredo Merial Inc, 3239 Satelite Blvd, Duluth, GA 30096.

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Abstract

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs.

ANIMALS 22 purpose-bred Beagles.

PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination.

RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs.

CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Abstract

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs.

ANIMALS 22 purpose-bred Beagles.

PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination.

RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs.

CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Osteoarthritis is a degenerative disease of humans and other animals that results in substantial medical problems.1–3 As disease develops, damage to articular cartilage exposes subchondral bone, resulting in synovial inflammation, pain, swelling, and often loss of mobility. Strong correlations exist between osteoarthritis and age, obesity, and excessive use of joints.1–3 In dogs, osteoarthritis is one of the most common causes of lameness and is estimated to affect approximately 20% of dogs > 1 year of age.4,5

Because no curative treatment is available for osteoarthritis, current pharmacological approaches primarily involve alleviation of pain and reduction of inflammation.1–3 In humans and horses with osteoarthritis and in dogs with experimentally induced disease, reductions in the severity of symptoms or clinical signs have been obtained with intra-articular injections of HA.6–10 Similarly, intra-articular or IM injections of polysulfated glycosaminoglycans and orally administered glucosamine and chondroitin sulfate have been associated with temporary resolution of clinical signs, although the underlying mechanisms of these therapeutic approaches remain to be fully elucidated.1 Collectively, these approaches have only limited efficacy and the drugs must be administered frequently owing to their rapid clearance.11 Frequent intra-articular injections are laborious, pose a risk of infection, cause stress, and are expensive. Hence, a clear medical need remains for more efficacious and sustained treatments that are also cost effective.

Recombinant adeno-associated viral vectors offer a novel way to deliver therapeutic genes into cells within an injured site, and the genes then provide stable, in situ production of therapeutic agents at the vector transduction site. For diseases affecting joints, the intra-articular administration route is an attractive way to gain access to articular cartilage cells lacking a vascular supply. Intra-articular injections of rAAV vectors have resulted in successful transfer of genes into the cartilage and synovium in animals with experimentally induced rheumatoid arthritis and osteoarthritis.12–22 Furthermore, expression of various anti-inflammatory and anabolic growth factors, including interleukin-1 receptor antagonist, tumor necrosis factor-α antagonists, and insulin-like growth factor-1, has been identified after intra-articular injection of rAAV vectors or by ex vivo treatment of cells.20–24 Studies20,21 involving equine joints have revealed the ability of rAAV vectors encoding interleukin-1 receptor antagonist to provide long-term expression after vector administration only once. Furthermore, the safety of rAAV-mediated gene transfer has been demonstrated in arthritic joints of humans and nonhuman primates and in healthy joints of horses.20–22,24–26

Hyaluronic acid, a polymer composed of glucuronic acid and N-acetyl glycosamine, is a potential multifunctional therapeutic agent owing to its pain-relieving, lubricating, and disease-modifying properties.6–10 In healthy joints, HA is generated by HASs expressed in fibroblast-like cells in the synovial lining and cartilage chondrocytes. The chondrocyte-derived HA forms a backbone to which proteoglycans attach. This proteoglycan-HA structure interweaves with collagen to provide a protective load-bearing surface over the underlying subchondral bone. Conversely, the lubrication properties of the synovial fluid are provided by HA synthesized by the fibroblast-like cells in the synovial lining.

The HAS enzyme exists in 3 isoforms, and the HAS2 form synthesizes the high-molecular-weight HA (> 2 × 106 Da) that is responsible for the viscosity of synovial fluid.27–31 Consequently, decreases in expression of the HAS2 gene as well as in the concentration and molecular weight of HA have been identified in synovial fluids obtained from humans and dogs with osteoarthritis.4,29 The importance of HAS2 in healthy cartilage was demonstrated in a study32 in which HAS2 activity was inhibited in chondrocytes in vitro. Not only did this intervention reduce HA accumulation and cell-associated matrix assembly, but it also caused an increase in the release of newly synthesized proteoglycan. Thus, the production of HA by the HAS2 isoform in cartilage and synovial tissues plays an important role in joint health.

The purpose of the study reported here was to evaluate the potential usefulness of rAAV vectors with AAV2 or AAV5 serotype capsid and expressing cHAS2 for enhancement of HA synthesis and therefore treatment of osteoarthritis in dogs. We also sought to determine the safety of local cHAS2 overexpression in the joints of healthy dogs as well as efficiency and localization of the rAAV-mediated gene transfer to various joint cells. To our knowledge, this study would represent the first evaluation of rAAV-mediated delivery of the gene for cHAS2 into joints and assessment of local HA production in a large (stifle) joint in dogs.

Materials and Methods

Vector generation

A plasmid expression cassette with a CBA promoter driving cHAS2 expression was generated to evaluate the feasibility of increasing HA production in cells. The cHAS2 expression cassette was subsequently incorporated into rAAV vectors with either AAV2 or AAV5 serotype capsid (Figure 1).

Figure 1—
Figure 1—

Diagrams of cHAS2 expression cassettes in plasmid vectors (A) and rAAV viral vectors (B) assessed in vitro for the concentration and size of secreted HA. cHASco = Codon-optimized cHAS cDNA. HI = Hybrid intron. pA = BGHpA.

Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.505

To achieve these aims, the sequence of the canine HAS2 gene (Genbank accession No. XM 539153.3) was codon-optimizeda for expression in dogs. The codon-optimized cHAS2 cDNA (1,656 bp) was synthesized with flanking NheI-NsiI restriction enzyme sites. This fragment was cloned into a plasmid containing ubiquitous CBA promoter, a hybrid intron (CBA and rabbit β-globin intron), and a BGHpA to generate pCBA-HI-cHAS2-BGHpA.

To generate rAAV vectors, the CBA-HI-cHAS2-BGHpA expression cassette was cloned between the AAV ITRs in a plasmid to generate psITR/CBA-HI-cHAS2-BGHpA. A 600-bp stuffer DNA (chromosome 16 P1 clone 96.4B) was included upstream of the expression cassette to generate a viral vector genome of 4,500 bp total. The rAAV vectors were generated by triple transfection of psITR/CBA-HI-cHAS2-BGHpA, pIM45BD rep-cap plasmid (AAV2 vectors) or pHLP19-cap5 (AAV5 vectors); pAdHELP and the vectors were purified by CsCl gradient. Vector yields were quantified with a real-time qPCR assayb by use of primers and a probe specific to the polyA sequences of bovine growth hormone and a standard curve of serially diluted linearized plasmid DNA containing BGHpA. The sequences used were as follows: forward primer, 5′-TCTAGTTGCCAGCCATCTGTTGT-3′; reverse primer, 5′-TGGGAGTGGCACCTTCCA-3′; and probe, 5′-TCCCCCGTGCCTTCCTTGACC-3′. The rAAV yields were expressed as vector genomes per milliliter.

In vitro assays

Plasmid vectors encoding cHAS2 or EGFP were transfectedc into HEK cellsd in serum-free mediume or medium containing 10% serum.f The rAAV vectors were incubated overnight with HEK cells at various concentrations of vector genomes per cell, the medium was changed, and the conditioned medium was collected 72 hours later. Concentrations of HA in collected medium were determined with the aid of a commercial HA test kit.g

The molecular weight of HA was assessed by agarose gel electrophoresis of concentrated conditioned medium; HA size markersh were run in parallel. An identical set of samples was digested with hyaluronidase at 37°C for 2 hours followed by 95°C for 5 minutes and then run on an agarose gel. Both gels were stained with cationic carbocyanine multipurpose dyei overnight followed by destaining in water.

In vivo evaluation

Protocol—All procedures involving animals were approved by the Preclinical Research Services Institutional Animal Care and Use Committee. Thirty purpose-bred 8-month-old Beagles (males and females; body weight, 6.6 to 8.8 kg) were acquired from a providerj and prescreened for serum antibody against AAV2 and AAV5 capsids. Of these, 22 dogs with a serum titer ≤ 4 for neutralizing antibodies against both capsids were used in the study. The random number function in a commercial spreadsheet programk was used to randomly allocate dogs on the basis of preshipment body weights to 5 treatment groups by assigning a random number to each dog and sorting these data from lowest to highest number. This was performed for each sex to allow an approximately equal allocation per group. The 5 test treatments included PBS solution (n = 2); rAAV2-cHAS2 at 1 × 1011 vector genomes/joint (low dose; 5), 5 × 1011 vector genomes/joint (medium dose; 5), or 10 × 1011 vector genomes/joint (high dose; 5); or rAAV5-cHAS2 at 5 × 1011 vector genomes/joint (medium dose; 5).

In preparation for treatment administration, dogs were premedicated with acepromazine maleate, morphine, and atropine sulfate, and anesthesia was induced and maintained with isoflurane in oxygen. All test treatments were administered as a 1-mL injection via a 25-gauge needle into the joint space of left stifle joint on day 0. The needle was removed, the injection site was pressed for 15 seconds, and the joint was extended and flexed 5 times to distribute the assigned test treatment. All dogs were subsequently examined twice daily for the next 7 days and then once daily for the remainder of the 28-day study period.

Sample collection—Blood samples were collected 1 week before (day -7) and 1, 14, and 28 days after treatment administration for WBC counts and for serum harvest to determine titers of neutralizing antibodies against viral capsids on days -7, 14, and 28. Synovial fluid samples were collected from sedated dogs on days -7 and 28 for quantification of HA concentration. Synovial fluid samples obtained on day 28 were also evaluated for titers of neutralizing antibodies against AAV capsids. For dogs in the PBS solution group, samples were collected from the left stifle joint only, and for dogs in the remaining groups, samples were collected from both stifle joints.

At the end of the 28-day study period, all dogs were euthanized by injection of a pentobarbital-based solution. Samples of synovium and cartilage were obtained from the lateral aspect of the joint and femoral condyle and tibial plateau (Figure 2), respectively, for qPCR analyses (to confirm viral vector uptake into synovial cells). For samples intended for qPCR analysis, the surgical instruments were changed between each dog and tissue sample. These samples were stored frozen at −80°C until processed for isolation of DNA and RNA.

Figure 2—
Figure 2—

Diagrams showing the locations (circled regions) within the stifle joint where synovium samples (A) and cartilage samples (B) were collected from healthy 8-month-old Beagles that received an intra-articular injection of PBS solution (n = 2); rAAV2-cHAS2 at 1 × 1011 vector genomes/joint (5), 5 × 1011 vector genomes/joint (5), or 10 × 1011 vector genomes/joint (5); or rAAV5-cHAS2 at 5 × 1011 vector genomes/joint (5). In panel A, location A is near the injection site, whereas location B is distant from the injection site.

Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.505

Tissue analyses—For DNA and RNA isolation and qPCR analysis to confirm the presence of viral genomes in rAAV-injected joints and determine the extent of dispersal, frozen tissue sections from synovium and cartilage samples were homogenized in 1 mL of bufferl containing 10 μL of β-mercaptoethanol and 1-mm zirconia beads m for 1 minute (2 to 3 times) with a cell disruptor.n A portion of the homogenate was transferred into phenol and guanidium isothiocyanate-based RNA-DNA-protein extraction solutiono (for RNA) or phenol-free cell distruption reagentp (for DNA).

The DNA was isolated in accordance with the instructions provided with the reagentp up to the aqueous phase, after which the samples were treated with RNase and the DNA was purified by use of DNA-binding columnsq in accordance with the manufacturer's protocol. The quality of DNA was assessed by assessment of the ratio of absorbance at 260 and 280 nm and agarose gel analysis. For RNA isolation, the homogenate was processed in accordance with the manufacturer's protocol, followed by RNA-binding columns.r The integrity of the RNA was assessed with the aid of a microfluidics-based nucleic acid analyzer,s and the ratio of absorbance at 260 and 280 nm. The cDNA was generated by use of a commercial kit.t Vector genome and HAS2 mRNA copies in tissue samples were quantified via qPCR assay with BGHpA-specific probes and primers as described for rAAV vector quantification; copies were expressed as vector genomes per cell (by use of 5 pg of double-stranded DNA/diploid cellular genome).

For histologic analysis, the medial aspect of each joint was fixed for 2 days in neutral-buffered 10% formalin and decalcified in 10% formic acid for up to 3 weeks. The trimmed medial aspect of the stifle joint was embedded in paraffin and sectioned. Eight-micrometer sections were stained with toluidine blue and examined by a board-certified veterinary pathologist. Cartilage samples were evaluated for severity of cartilage lesions and proteoglycan loss (0 = normal; 5 = severe), and synovium samples were evaluated for density of inflammatory cells (0 = normal; 5 = severe).19

Neutralizing antibody quantitation—Titers of neutralizing antibodies against AAV5 and AAV2 capsids in serum and synovial fluid samples were determined by use of a cell-based bioassay as described elsewhere,33 with slight modifications. Recombinant AAV2 and rAAV5 vectors with the Escherichia coli LacZ gene, encoding β-galactosidase, were used in the assays. The vectors were generated by triple transfection and purified by CsCl gradient. Serum samples were heat-inactivated at 56°C for 30 minutes before testing. Briefly, HeLa cellsu were plated at 20,000 cells/well in 96-well culture plates. The following day, the cells were infected with temperature-sensitive adenovirus 5 mutant (Ad5ts149) at a multiplicity of infection of 0.5 for rAAV2 or 2 for rAAV5 for 4 hours at 39°C. The serum and synovial fluid samples were first diluted 1:2 and then serially diluted 2-fold in triplicate across 96-well plates. An equal volume of rAAV2-LacZ or rAAV5-LacZ (1,000 vector genomes/cell) was added to the diluted samples, after which they were incubated for 1 hour at 37°C. After cell infection with Ad5ts149, medium was removed from cells and replaced with 150 μL of fresh medium. Fifty microliters of the test sample-AAV mixture was added to cells and incubated at 39°C for 3 days. Cells were assayed for β-galactosidase activity with a chemiluminescent β-galactosidase assay kitv in accordance with the manufacturer's instructions, and then signal detection was performed with a microplate reader. Neutralizing antibody titer was defined as the reciprocal of the highest dilution that inhibited viral transduction (β-galactosidase activity) by ≥ 50%, compared to cells infected in the absence of serum or synovial fluid. A value < 4 was considered a negative result.

Statistical analysis

Summary statistics were computed, and data were reported as mean ± SD. No statistical tests were performed.

Results

In vitro evaluation of cHAS2 expression vectors

Concentrations of HA in culture medium from HEK cells transfected with plasmid encoding cHAS2 were 6.5- and 9-fold as high as concentrations in culture medium from untransfected and negative control plasmid (EGFP) transfected cells, respectively (Figure 3). High-molecular-weight HA was detected in the culture medium from cells transfected with the HAS2 expression plasmid. The secreted product was larger than 1.5 MDa (as estimated by use of HA molecular-weight markers) and was no longer detectable after digestion with hyaluronidase, indicating that the product was HA. These data were interpreted as indicating that transfer of the gene for cHAS2 to HEK cells increased production and secretion of high-molecular-weight HA in vitro.

Figure 3—
Figure 3—

Characterization of plasmid and viral vectors with the cHAS2 expression cassette in Figure 1. A—Mean concentrations of HA secreted by cells transfected with the cHAS2 expression plasmid or an EGFP expression plasmid (negative control treatment) and untransfected cells. Plasmids were transfected into HEK cells, and medium (serum-free medium [dark gray bars] or medium with 10% serum [light gray bars]) was harvested 3 days later. The HA concentrations were quantitated by means of an HA-binding protein-based detection system. B—Photographs of electrophoretic gels showing the molecular weight of HA (size markers indicated) following transfection with the cHAS2 expression plasmid. For this analysis, conditioned medium (CM) samples were subjected to agarose gel electrophoresis, followed by staining with a multipurpose dye or to digestion first with hyaluronidase for 2 hours, followed by gel analysis and staining. HMW = High molecular weight. A 1.6-MDa HA marker was also run at 3 concentrations (10, 2, and 0.2 μg/lane). C—Mean concentrations of HA in conditioned medium as secreted by HEK cells in vitro, suggesting the potency of the infecting vector. For these assays, HEK cells were infected with various concentrations of AAV2-cHAS2 vector or AAV2-EGFP vector or left uninfected, and HA concentrations were quantitated 3 days later. vg = Vector genome. D—Same results as shown in panel C, except involving greater virus doses and 2 lots (instead of 1 lot) of AAV5-HAS2 vector. In panels A, C, and D, values represent the mean of 2 to 3 transfections/group and assays were performed a minimum of 2 times; error bars represent SD.

Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.505

Infection with either vector rAAV2 or rAAV5 resulted in a dose-responsive increase in the concentration of HA in the culture medium (Figure 3). Because of the low infection rate of the HEK cells by AAV5 in vitro, greater concentrations of the rAAV5 vectors were required to achieve this result.

In vivo evaluation of rAAV-cHAS2 viral vectors

No adverse clinical signs, body weight changes, lameness, or death were observed in any of the 22 healthy Beagles during the study. Blood WBC counts were within the reference range at all assessment points, except for 2 dogs prior to treatment, which had a high WBC count on day -7 and then an unremarkable count for the remainder of the study (data not shown).

Serum titers of neutralizing antibodies against AAV capsids at various points were summarized for individual dogs (Table 1). Serum neutralizing antibody titers were higher for AAV5 capsid than for AAV2 capsid and increased from day 14 to day 28. Such neutralizing antibodies were also detected in the synovial fluid samples obtained on day 28 from all rAAV-injected joints (Table 2). Furthermore, synovial fluid titers of neutralizing antibody against AAV2 capsids were comparable with corresponding serum titers, whereas the synovial fluid titers against AAV5 capsid were higher than titers in the matched serum samples. Neutralizing antibodies against these capsids were also detected in synovial fluid samples obtained from the contralateral uninjected joint, but titers were consistently lower than the titers in synovial fluid samples obtained from the vector-injected joints.

Table 1—

Serum titers of neutralizing antibodies against AAV2 and AAV5 capsids at various points for healthy Beagles (12 males and 10 females; body weight, 6.6 to 8.8 kg) that received an intra-articular injection into the left stifle joint of PBS solution (n = 2); rAAV2-cHAS2 at 1 × 1011 vector genomes/joint (low dose; 5), 5 × 1011 vector genomes/joint (medium dose; 5), or 10 × 1011 vector genomes/joint (high dose; 5); or rAAV5-cHAS2 at 5 × 1011 vector genomes/joint (medium dose; 5) on day 0.

  AAV2AAV5
TreatmentDog No.Day -7Day 14Day 28Day -7Day 14Day 28
PBS solution1< 4< 4< 4< 444
 2< 4< 4< 4< 4< 4< 4
Low-dose rAAV234256128< 4NTNT
 4< 42,0482,048< 4NTNT
 5< 44,0962,048< 4NTNT
 6< 464512< 4NTNT
 74128512< 4NTNT
Medium-dose rAAV28< 42,0481,024< 4NTNT
 9< 42,0482,048< 4NTNT
 10< 41,0241,024< 4NTNT
 11< 45122,048< 4NTNT
 1242,0481,0244NTNT
High-dose rAAV213< 42,0481,024< 4NTNT
 14< 42,0481,024< 4NTNT
 15< 41,0241,024< 4NTNT
 16< 44,0964,096< 4NTNT
 1742,0482,0484NTNT
Medium-dose rAAV518< 4NTNT< 48,1928,192
 19< 4NTNT< 42,04816,384
 20< 4NTNT< 44,09616,384
 21< 4NTNT42,04816,384
 22< 4NTNT< 44,09616,384

NT = Not tested.

Table 2—

Serum and synovial fluid titers of neutralizing antibodies against AAV2 and AAV5 capsids for healthy Beagles 28 days following intra-articular injection into the left stifle joint of a medium dose of rAAV2-cHAS2 (5 × 1011 vector genomes/joint; n = 5) or rAAV5-cHAS2 (5 × 1011 vector genomes/joint; 5).

TreatmentDog No.SerumSynovial fluid from rAAV-injected (left) jointSynovial fluid from uninjected (right) joint
rAAV281,0241,02464
 92,0484,096256
 101,0244,096256
 112,0482,04864
 121,02425632
rAAV5188,192131,0724,096*
 1916,384131,072512
 2016,38465,53616,384
 2116,38465,5368,192
 2216,38465,5364,096*

All values represent reciprocals of serum or synovial fluid dilutions.

Sample diluted because of a low volume of synovial fluid recovered.

Histologic examination of cartilage samples from the vector-injected joints revealed minimal proteoglycan loss and cartilage degeneration, with mean ± SD scores for injected (left) and uninjected (right) stifle joints, respectively, appearing similar as follows: low-dose rAAV2, 0.3 ± 0.4 and 0.3 ± 0.2; medium-dose rAAV2, 0.4 ± 0.2 and 0.5 ± 0.3; high-dose rAAV2, 0.4 ± 0.4 and 0.4 ± 0.2; and medium-dose rAAV5, 0.6 ± 0.2 and 0.7 ± 0.2. These findings, in addition to those for PBS solution-injected joints (mean ± SD score, 0.5 ± 0.0), suggested that the observed changes were typical age-related changes rather than associated with the intra-articular injections.

Similarly, histologic examination of synovium samples, regardless of treatment or lack of treatment, revealed few changes, with mean ± SD scores of 0.0 ± 0.0 for all joints except those injected with medium-dose rAAV5 (1.2 ± 0.4). Minimal to mild synovitis extending into the joint capsule and medial collateral ligament was observed in all stifle joints injected with rAAV5 vector; these findings were not observed in the corresponding uninjected joint. Overall, administration of both rAAV2 and rAAV5 vectors appeared well tolerated from a histologic perspective.

Vector DNA was detected in the synovium samples collected close to the injection site (location A) in most rAAV-injected joints (Figure 4). Synovium samples from rAAV2-cHAS2-injected joints contained a mean of 0.42 ± 0.57 vector genomes/cell, and there was no clear difference in the concentration of vector DNA detected between low-and high-dose rAAV2-injected joints. Synovium samples from rAAV5-cHAS2-injected joints consistently had higher amounts of vector DNA, ranging from 1 to 12 vector genomes/cell. Low amounts of vector DNA were detected in some contralateral (uninjected) joints and, particularly, in the low-dose rAAV2-injected joints (data not shown). Vector DNA was detected more consistently in synovium samples obtained away from the injection site (location B; showing dispersal) in low-dose rAAV2-injected joints, and values for those joints were comparable with those measured in the samples collected close to the injection site (location A). No vector DNA was detected in synovium samples from location B in high-dose rAAV2-injected joints. In contrast, all synovium samples from location B in medium-dose rAAV5-cHAS2-injected joints had detectable vector DNA; however, the amount of vector DNA in these samples was 10-fold less than the amount detected in samples obtained from location A, suggesting a location-dependent transduction efficiency.

Figure 4—
Figure 4—

Results of rAAV vector genome and cHAS mRNA detection in synovium samples obtained from the treated left stifle joint of 8-month-old Beagles (12 males and 10 females; body weight, 6.6 to 8.8 kg) that received an intra-articular injection of PBS solution (n = 2); rAAV2-cHAS2 at 1 × 1011 vector genomes/joint (low dose [L]; 5), 5 × 1011 vector genomes/joint (medium dose [M]; 5), or 10 × 1011 vector genomes/joint (high dose [H]; 5); or rAAV5-cHAS2 at 5 × 1011 vector genomes/joint (medium dose [M]; 5) and were euthanized for sample collection 28 days later. A—Number of vector genome DNA and cHAS mRNA copies per synovial cell by dog (individually numbered). *Dog had a serum titer of 4 for neutralizing antibodies against both AAV2 and AAV5 capsids. B—Mean number of vector genome DNA and cHAS mRNA copies per synovial cell by treatment group for synovium samples collected from location A in Figure 2. C—Mean number of vector genome DNA and cHAS mRNA copies per synovial cell by treatment group for synovium samples collected from location B in Figure 2. All tissue samples were collected 28 days after treatment injection and analyzed by means of qPCR assay to detect BGHpA. In panels B and C, error bars represent SD.

Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.505

Copies of vector-derived cHAS2 mRNA were detected in synovium samples from location A in 1, 3, and 3 of 5 joints injected with low-, medium-, and high-dose rAAV2, respectively, whereas vector-derived mRNA was detected in synovium samples from all 5 rAAV5-cHAS2-injected joints (Figure 4). Although cHAS2 mRNA from the rAAV5 vector was also detected in the synovium samples from location B, numbers of mRNA copies were lower than detected in the synovium samples from location A. This finding was consistent with the reduced detection of vector DNA. Overall, the detection of cHAS2 transcripts correlated well with the presence of vector DNA; all synovium samples containing at least 0.1 vector genomes/cell also contained vector-derived mRNA.

Results for cartilage samples obtained from the femoral condyle of each injected joint and means for each treatment group were graphically displayed (Figure 5). Detection of the rAAV2-cHAS2 genome was sporadic, and there was little evidence of a dose-related response. Vector-derived transcripts were detected in only 1 rAAV2-cHAS2-injected joint. In contrast, vector genomes were consistently detected in all femoral cartilage samples from rAAV5-cHAS2-injected joints, and each tested sample also contained cHAS2 transcripts (1 dog was not tested for expression). For cartilage samples obtained from the tibial plateau, no vector DNA was detected in any of the rAAV2-cHAS2-injected joints, whereas both vector DNA and cHAS2 transcripts were detected in all samples collected from the rAAV5-cHAS2-injected joints. No vector DNA was detected in any cartilage sample from the femoral condyle or tibial plateau of the contralateral, uninjected joints (data not shown).

Figure 5—
Figure 5—

Results of rAAV vector genome and cHAS mRNA detection in stifle joint cartilage samples obtained from the treated left stifle joint of the dogs in Figure 4 following euthanasia on day 28. A—Number of vector genome DNA and vector-derived mRNA copies detected per chondrocyte for femoral condyle cartilage samples by dog (individually numbered). B—Mean number of vector genome DNA and mRNA copies per chondrocyte by treatment group for femoral condyle cartilage samples. C—Mean number of vector genome DNA and mRNA copies per chondrocyte by treatment group for tibial plateau cartilage samples. See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.505

Results of vector DNA detection in cartilage and synovium samples from injected joints were graphically displayed (Figure 6). Higher and more consistent gene transfer was achieved with the rAAV5 vector than with the rAAV2 vector. Gene transfer to cartilage was similar between rAAV5-cHAS2 and rAAV2-cHAS2 vectors for femoral condyle samples, whereas only the rAAV5 vector genomes were detected in tibial plateau samples. Furthermore, only the rAAV5 vector resulted in a consistent expression of the vector-derived cHAS2 mRNA in all tested anatomic locations.

Figure 6—
Figure 6—

Results of rAAV vector genome and cHAS mRNA detection in stifle joint synovium and cartilage samples obtained from the treated left stifle joint of the dogs in Figure 4 following euthanasia on day 28. A—Mean number of vector genome copies per cell in synovium samples from locations A and B in Figure 2 and in femoral condyle (FC) and tibial plateau (TP) cartilage samples by treatment group. B—Mean number of rAAV5-cHAS2 vector DNA copies and cHAS2 mRNA (vector RNA) copies in synovium or cartilage samples. See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.505

Concentrations of HA in synovial fluid samples were generally increased from baseline values (as shown by normalized values) with high-dose rAAV2-cHAS2 and medium-dose rAAV5-cHAS2 (Figure 7). Furthermore, the increase in HA concentration in the high-dose rAAV2-injected joints generally correlated to the cHAS2 mRNA detection values for synovium samples collected from location A of injected joints, given that no transcripts were detected in any other anatomic locations tested with this treatment group. In the remaining treatment groups, synovial fluid HA concentrations remained unchanged or were lower than baseline on day 28.

Figure 7—
Figure 7—

Mean percentage change from baseline (day -7) in HA concentrations in synovial fluid from the treated (left) stifle joint at 4 weeks following treatment (day 28) by treatment group (A) and HA concentrations at days -7 and 28 for the individual dogs (B) in Figure 4. Arrows indicate dogs with higher synovial fluid HA concentrations after versus before treatment. See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.505

Discussion

Local delivery of a therapeutic agent into an osteoarthritic joint is an attractive treatment strategy for osteoarthritis that minimizes the potential for adverse systemic effects. In the present study, we generated rAAV vectors with cHAS2 expression cassettes and then performed experiments to determine whether production of HA could be increased in treated joints as a potential means of providing local pain relief and disease-modifying and lubrication properties for dogs with osteoarthritis. The in vitro findings corroborated those in a previous report28 that delivery of the gene for cHAS2 increases secretion of high-molecular-weight HA by HEK cells. Furthermore, our in vivo data indicated that the delivery of the rAAV vectors encoding cHAS2 into the joints of healthy dogs facilitated gene transfer into both synovial and cartilage tissues in a capsid-dependent manner and that overexpression of HA in canine joints did not cause adverse effects.

The AAV capsid serotypes AAV2 and AAV5 were chosen for evaluation because of their reported safety for providing gene transfer to joints of humans, horses, and nonhuman primates.20–22,24–28 The selection of these AAV capsid serotypes was important because preexisting immunity to capsid in the target species can result in clearance of the vector and prevention of gene transfer. Preexisting neutralizing antibodies against various AAV capsid serotypes have been detected in the sera of humans and other animals, including dogs and horses.34–36 Similarly, neutralizing antibodies have been detected in synovial fluids of humans with rheumatoid arthritis, although the titers are lower than those in matched serum samples.35,37,38

Serologic testing of 28 of the 30 dogs screened for inclusion in the present study yielded no or low titers of neutralizing antibody against AAV2 or AAV5 capsids. Five dogs with borderline positive titers (ie, a value of 4) against AAV2 or AAV5 capsids had numbers of vector genomes in synovial tissues comparable with those of dogs with undetectable titers. Because seropositive dogs were excluded from the study, it remains unknown whether any neutralizing antibodies in synovial fluid would have rendered the gene transfer ineffective. Overall, our data regarding preexisting humoral immunity to AAV2 and AAV5 capsids in dogs is in agreement with published data.34,36 As expected, serum titers of neutralizing antibodies against capsids increased in all dogs after intra-articular administration of rAAV vectors, with a general dose-related response observed in dogs treated with rAAV2 and higher titers observed against AAV5 capsid. Indeed, 1 dog in the high-dose rAAV2 group that lacked any evidence of gene transfer in any analyzed tissues had a relatively high titer against AAV2 capsid.

Dose-dependent increases in titers of neutralizing antibodies against AAV capsids have been identified in nonhuman primates and humans after intra-articular rAAV5 and rAAV2 injection, respectively.24,26 In the present study, neutralizing antibodies against AAV2 and AAV5 capsids were detected in synovial fluid samples from both vector-injected and corresponding uninjected joints after vector delivery. These titers were considerably higher in the vector-injected versus uninjected joints, which is similar to reported findings for horses.39 The reason synovial fluid titers against AAV5 capsid were numerically higher than those against AAV2 capsid remains unclear. However, efficient transduction of murine dendritic cells, T cells, and macrophages by rAAV5 vectors but not by AAV2 has been reported.40 Overall, these data suggested that subsequent delivery of the same vector to a previously treated joint or uninjected joint in the study dogs would likely have been challenging. However, successful repeated joint injection with a different AAV serotype vector has been demonstrated in horses.21

The serotype of AAV capsid determines vector tropism and the efficiency of vector uptake by target cells. Although primary AAV receptors are typically extracellular carbohydrates, the secondary receptors are usually cell-surface proteins.41,42 For AAV2, the primary receptor is heparan sulfate, which is abundant in the extracellular matrix of cartilage. Multiple coreceptors have been identified for AAV2, including human fibroblast growth factor receptor, hepatocyte growth factor receptor, laminin receptor, and integrin α-V β-5.41,42 For AAV5, known receptors include α-2,3-N-linked sialic acid and platelet-derived growth factor receptor.41,42 Platelet-derived growth factor receptors are present in chondrocytes and synoviocytes, and receptor expression increases during inflammation.43

The selection of an optimal AAV capsid for a particular target animal species is affected by species differences in expression of AAV receptors in vivo.13,18,19,44 Similarly, receptor expression in cells from target species in vitro is influenced by various cell culture conditions, such as the growth medium, oxygen concentration, cell density, or culture system (monolayer cultured cells vs tissue explant cultures) used.22,35,37,43,44 To avoid these potential difficulties, we tested rAAV2 and rAAV5 vector-mediated gene transfer directly in the target species and location in vivo, namely the stifle joint of dogs, after intra-articular injection. Because HA expression was desirable in both synoviocytes and chondrocytes, vector genomes were quantified in synovium and cartilage samples. Results indicated inconsistent gene transfer by rAAV2 vector to the tissues and a lack of a dose-response effect. These findings were unexpected, primarily because we have previously demonstrated consistent rAAV2 vector genome detection in both synovium and cartilage in experiments involving osteoarthritic rabbit joints with a comparable vector dose.19 In contrast, rAAV5 vector genomes were detected in a consistent manner in all cartilage and synovium samples in the present study.

Efficient rAAV5 vector uptake into the synovium has been demonstrated in joints of rodents and nonhuman primates.12,13,25 However, detection of rAAV5 genomes in canine cartilage was surprising given that healthy cartilage is reportedly difficult to infect in vivo owing to its extensive extracellular matrix. We are unaware of other reports on transduction of cartilage by rAAV5 in large animals. Transduction of chondrocytes by rAAV2 has been demonstrated in equine joints.20–22 Vectors taken into chondrocytes could provide more sustained expression of HA than those taken into synoviocytes owing to the limited proliferation of chondrocytes.20,21 Overall, these findings highlight species-specific differences in tissue tropism by various AAV capsids. Currently, rAAV5-based vectors are being developed for the treatment of rheumatoid arthritis in humans, and rAAV2 capsid and its variants have promise for the treatment of osteoarthritis in horses.20–22,25,26

In the study reported here, we generated a canine version of the HAS2 protein to minimize immune responses, although the human and canine amino acid sequences for HAS2 differ by only 2 amino acids. The CBA promoter was chosen for cHAS2 expression to provide sustained HA production in osteoarthritic joints, given that endogenous HAS2 expression has been reported to be downregulated by various inflammatory mediators.30,45,46 In equine synoviocytes and chondrocytes in vitro, the CBA and cytomegalovirus promoters result in comparable expression of vector-derived product.20 In the present study, the AAV-cHAS2 vector-derived gene expression from the CBA promoter was confirmed in canine synovial cells and chondrocytes. Expression of cHAS2 mRNA was detected more consistently when the rAAV5 vector was used than when the rAAV2 vector was used. This finding correlated with the increased detection of rAAV5 vector genome. Detection of HAS2 transcripts from rAAV5 vector in the cartilage samples confirmed that chondrocytes had been transduced rather than the virus having become sequestered in the extracellular matrix. For rAAV2, comparable amounts of vector genomes and transcripts were also detected in synovium samples. Because cHAS2 mRNA from AAV2 vectors was mostly undetectable in chondrocytes, the vector likely remained outside the chondrocytes, possibly retained within the extracellular matrix. The lack of detectable cHAS2 mRNA from AAV2 vectors could have been attributable to AAV2 capsids containing positively charged heparan-binding domains and the presence of heparan sulfate proteoglycans in articular cartilage. Alternatively, slow internalization of vector, uncoating of capsid, and expression kinetics reported for AAV2 may be responsible for the lower ratio of transcript to vector genome in cartilage, compared with the ratio for AAV5.47 Lastly, despite occasional detection of low amounts of vector genomes in contralateral uninjected joints, no cHAS2 transcripts were detected in any of these samples. Movement of rAAV vectors to the contralateral joints has been reported, and possible explanations include uptake and migration of lymphocytes and antigen-presenting cells via the bloodstream or lymphatics.16,26

Because the present study involved healthy dogs without osteoarthritis, no major structural changes in the cartilage were detected, as expected. In contrast, overexpression of cHAS2 in the synovium was expected to increase concentrations of HA in the synovial fluid; however, this was not a consistent finding. Only in some dogs in the high-dose rAAV2 and mediumdose rAAV5 treatment groups were posttreatment HA concentrations higher than pretreatment values. Because vector detection was inconsistent for the high-dose rAAV2 group, the HA data for this group may have suggested that the limited sample collection had resulted in missing the location for the rAAV2 vector transduction in the joint. In the remaining treatment groups, HA concentrations remained unchanged or even decreased following injection.

Differences exist in reported synovial fluid HA concentrations for healthy young dogs. In one report,4 values range from 1 to 3 mg/mL,4 whereas in another report,48 the mean concentration is 15.3 mg/mL.48 In the present study, synovial fluid HA concentrations varied from 1.3 to 18 mg/mL, and a similar range of values was obtained for another group of healthy dogs previously evaluated by our research group (data not shown). In contrast, dogs with severe osteoarthritis have a much lower synovial fluid HA concentration, ranging from approximately 0.2 to 2 mg/mL.4,48 Hence, any increase due to treatment may be easier to detect in osteoarthritic dogs. Evaluation of a higher vector dose for a longer period than used in the present study might help to validate the observed increase in HA production. Because the endogenous and rAAV-derived cHAS2 proteins were identical, we did not verify production of vector-derived cHAS2 protein in the joint tissues. Instead, the vector-derived DNA and cHAS2 mRNA were used as endpoints to confirm gene transfer and expression, respectively.

The results of the present study indicated that rAAV-mediated gene delivery of cHAS2 through intraarticular injection into the stifle joint was feasible and well tolerated in healthy, osteoarthritis-free dogs. Although preexisting serum titers of neutralizing antibodies against AAV2 and AAV5 capsids were low, the rAAV5 capsid vector appeared to provide higher and more consistent gene transfer and cHAS2 gene expression in synovial cells and chondrocytes than obtained with the rAAV2 capsid vector. Additional studies are required to determine whether rAAV5-mediated HAS2 gene transfer can effectively decrease the severity of joint lesions and reduce pain in dogs with osteoarthritis.

Acknowledgments

Supported by Merial Inc and Sanofi Co.

The authors report that there were no conflicts of interest.

The authors thank Alison Bendele for histologic analyses, Alison Schroeer for help with the illustrations, and James Moore for manuscript editing.

ABBREVIATIONS

AAV

Adeno-associated virus

BGHpA

Bovine growth hormone polyadenylation site

CBA

Chicken β-actin

cHAS2

Canine hyaluronic acid synthase-2

EGFP

Enhanced green fluorescent protein

HA

Hyaluronic acid

HAS

Hyaluronic acid synthase

HEK

Human embryonic kidney

ITR

Inverted terminal repeat

qPCR

Quantitative PCR

rAAV

Recombinant adeno-associated virus

Footnotes

a.

Thermo Fisher Scientific, Waltham, Mass.

b.

Applied Biosystems 7500 real-time PCR system, Thermo Fisher Scientific, Waltham, Mass.

c.

Lipofectamine 2000, Thermo Fisher Scientific, Waltham, Mass.

d.

AAV-293 cell line, Agilent Technologies, Santa Clara, Calif.

e.

Opti-MEM, Thermo Fisher Scientific, Waltham, Mass.

f.

High glucose DME, Irvine Scientific, Santa Ana, Calif.

g.

Corgenix Inc, Bloomfield, Colo.

h.

Select-HA HiLadder, Hyalose, Oklahoma City, Okla.

i.

Stains-All, Sigma-Aldrich Corp, St Louis, Mo.

j.

Marshall BioResources, North Rose, NY.

k.

Microsoft Excel, version 14.5, Microsoft Corp, Redmond, Wash.

l.

RLT Plus, Qiagen, Germantown, Md.

m.

Biospec Product Inc, Bartlesville, Okla.

n.

Beadbeater-16, Thermo Fisher Scientific, Waltham, Mass.

o.

TRIzol reagent, Tel-Test Inc, Friendswood, Tex.

p.

DNA STAT-60, Tel-Test Inc, Friendswood, Tex.

q.

PureLink column (Purelink Genomic DNA mini kit), Thermo Fisher Scientific, Waltham, Mass.

r.

TRIzol RNA binding columns, Promega Corp, Madison, Wis.

s.

Bioanalyzer, Agilent Technologies, Santa Clara, Calif.

t.

Applied Biosystem high capacity cDNA RT kit, Thermo Fisher Scientific, Waltham, Mass.

u.

American Type Culture Collection, Manassas, Va.

v.

Galacto-Star assay kit, Life Technologies, Carlsbad, Calif.

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  • Figure 1—

    Diagrams of cHAS2 expression cassettes in plasmid vectors (A) and rAAV viral vectors (B) assessed in vitro for the concentration and size of secreted HA. cHASco = Codon-optimized cHAS cDNA. HI = Hybrid intron. pA = BGHpA.

  • Figure 2—

    Diagrams showing the locations (circled regions) within the stifle joint where synovium samples (A) and cartilage samples (B) were collected from healthy 8-month-old Beagles that received an intra-articular injection of PBS solution (n = 2); rAAV2-cHAS2 at 1 × 1011 vector genomes/joint (5), 5 × 1011 vector genomes/joint (5), or 10 × 1011 vector genomes/joint (5); or rAAV5-cHAS2 at 5 × 1011 vector genomes/joint (5). In panel A, location A is near the injection site, whereas location B is distant from the injection site.

  • Figure 3—

    Characterization of plasmid and viral vectors with the cHAS2 expression cassette in Figure 1. A—Mean concentrations of HA secreted by cells transfected with the cHAS2 expression plasmid or an EGFP expression plasmid (negative control treatment) and untransfected cells. Plasmids were transfected into HEK cells, and medium (serum-free medium [dark gray bars] or medium with 10% serum [light gray bars]) was harvested 3 days later. The HA concentrations were quantitated by means of an HA-binding protein-based detection system. B—Photographs of electrophoretic gels showing the molecular weight of HA (size markers indicated) following transfection with the cHAS2 expression plasmid. For this analysis, conditioned medium (CM) samples were subjected to agarose gel electrophoresis, followed by staining with a multipurpose dye or to digestion first with hyaluronidase for 2 hours, followed by gel analysis and staining. HMW = High molecular weight. A 1.6-MDa HA marker was also run at 3 concentrations (10, 2, and 0.2 μg/lane). C—Mean concentrations of HA in conditioned medium as secreted by HEK cells in vitro, suggesting the potency of the infecting vector. For these assays, HEK cells were infected with various concentrations of AAV2-cHAS2 vector or AAV2-EGFP vector or left uninfected, and HA concentrations were quantitated 3 days later. vg = Vector genome. D—Same results as shown in panel C, except involving greater virus doses and 2 lots (instead of 1 lot) of AAV5-HAS2 vector. In panels A, C, and D, values represent the mean of 2 to 3 transfections/group and assays were performed a minimum of 2 times; error bars represent SD.

  • Figure 4—

    Results of rAAV vector genome and cHAS mRNA detection in synovium samples obtained from the treated left stifle joint of 8-month-old Beagles (12 males and 10 females; body weight, 6.6 to 8.8 kg) that received an intra-articular injection of PBS solution (n = 2); rAAV2-cHAS2 at 1 × 1011 vector genomes/joint (low dose [L]; 5), 5 × 1011 vector genomes/joint (medium dose [M]; 5), or 10 × 1011 vector genomes/joint (high dose [H]; 5); or rAAV5-cHAS2 at 5 × 1011 vector genomes/joint (medium dose [M]; 5) and were euthanized for sample collection 28 days later. A—Number of vector genome DNA and cHAS mRNA copies per synovial cell by dog (individually numbered). *Dog had a serum titer of 4 for neutralizing antibodies against both AAV2 and AAV5 capsids. B—Mean number of vector genome DNA and cHAS mRNA copies per synovial cell by treatment group for synovium samples collected from location A in Figure 2. C—Mean number of vector genome DNA and cHAS mRNA copies per synovial cell by treatment group for synovium samples collected from location B in Figure 2. All tissue samples were collected 28 days after treatment injection and analyzed by means of qPCR assay to detect BGHpA. In panels B and C, error bars represent SD.

  • Figure 5—

    Results of rAAV vector genome and cHAS mRNA detection in stifle joint cartilage samples obtained from the treated left stifle joint of the dogs in Figure 4 following euthanasia on day 28. A—Number of vector genome DNA and vector-derived mRNA copies detected per chondrocyte for femoral condyle cartilage samples by dog (individually numbered). B—Mean number of vector genome DNA and mRNA copies per chondrocyte by treatment group for femoral condyle cartilage samples. C—Mean number of vector genome DNA and mRNA copies per chondrocyte by treatment group for tibial plateau cartilage samples. See Figure 4 for remainder of key.

  • Figure 6—

    Results of rAAV vector genome and cHAS mRNA detection in stifle joint synovium and cartilage samples obtained from the treated left stifle joint of the dogs in Figure 4 following euthanasia on day 28. A—Mean number of vector genome copies per cell in synovium samples from locations A and B in Figure 2 and in femoral condyle (FC) and tibial plateau (TP) cartilage samples by treatment group. B—Mean number of rAAV5-cHAS2 vector DNA copies and cHAS2 mRNA (vector RNA) copies in synovium or cartilage samples. See Figure 4 for remainder of key.

  • Figure 7—

    Mean percentage change from baseline (day -7) in HA concentrations in synovial fluid from the treated (left) stifle joint at 4 weeks following treatment (day 28) by treatment group (A) and HA concentrations at days -7 and 28 for the individual dogs (B) in Figure 4. Arrows indicate dogs with higher synovial fluid HA concentrations after versus before treatment. See Figure 4 for remainder of key.

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