AAV-Based Gene Therapy

Abstract

The invention relates to the field of andeno-associated virus (AAV) based gene therapy, in particular to the use of a combination of recombinant AAV-transgene vectors with an immunosuppressant and/or empty-AAV capsids. The invention further provides a composition and a kit of parts based on this combination.

Claims

1. A rAAV vector composition and an immunosuppressant for use in a treatment comprising gene therapy, wherein the treatment comprises the administration of the rAAV vector composition and the administration of the immunosuppressant to an individual, wherein the rAAV vector composition comprises a rAAV-transgene vector and an empty capsid in a ratio of empty capsid to rAAV-transgene vector of at least 1:1.

2. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the at least one of the rAAV vector composition and immunosuppressant is administered locally.

3. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein at least one of the rAAV vector composition and the immunosuppressant is administered systemically.

4. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the rAAV vector composition and the immunosuppressant are administered sequentially.

5. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the immunosuppressant is an innate immune cell inhibitor, a cytostatic drug, a non-steroidal anti-inflammatory drug, and/or an immunosuppressant biological such as a macrophage depleting antibody, a TNF blocker, IL-6 blocker and/or an IL-2 blocker and/or a purinergic signaling pathway modifying drug.

6. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 5, wherein the immunosuppressant is an innate immune cell inhibitor such as glucocorticoid and/or a liposomal bisphosphonate.

7. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the transgene comprised in the rAAV-transgene vector encodes a therapeutic protein.

8. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the gene therapy is for preventing, delaying, curing, reverting and/or treating an inflammatory condition or inflammatory disease.

9. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 8, wherein the transgene encodes a therapeutic anti-inflammatory protein.

10. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 8, wherein the inflammatory condition or disease is a rheumatic condition or disease.

11. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 2, wherein the rAAV vector composition is administered intra-articularly.

12. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the gene therapy is for treating, preventing, delaying, curing, reverting and/or treating an non-inflammatory condition or non-inflammatory disease.

13. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the rAAV vector composition further comprises a pharmaceutically acceptable carrier, diluents, solubilizer, filler, preservative and/or excipient.

14. A rAAV vector composition and an immunosuppressant for use in a treatment according to claim 1, wherein the immunosuppressant is comprised within the rAAV vector composition.

15. A rAAV vector composition, wherein the immunosuppressant is comprised within the rAAV vector composition.

16. A kit of parts comprising: a rAAV vector composition comprises a rAAV-transgene vector and an empty capsid in a ratio of empty capsid to rAAV-transgene vector of at least 1:1 and; an immunosuppressant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1: Luciferase expression is influenced by vector administration before or after the onset of arthritis. After induction of arthritis, mice (n=5 per group) were injected with 1.5e10 vg of a rAAV5-transgene vector encoding the gene for luciferase (rAAV.CMV.Fluc5) intra-articularly either on day 17 before onset of arthritis or day 24 after onset of arthritis. Imaging was performed 3 days after vector injection and thereafter weekly up to 4 weeks (group day 24) or 5 weeks (group day 17). Inset: Percentage of knee joints expressing a positive signal. A positive signal was defined as a value 1.5 times above the upper limit of the value of control knee joints for at least one measurement in time. Luminescence is shown per group as average; error bars, SEM.

[0083] FIG. 2: Effect of addition of liposomal clodronate, triamcinolone and empty capsids on rAAV5-luciferase expression. After induction of arthritis, mice (n=5 per group) were injected with 1.5e10 vg of rAAV5.CMV.FLuc vector intra-articularly. Imaging was performed 3 days after vector injection and thereafter weekly up to 4 weeks. (a) Addition of liposomal clodronate (5 μl/g i.v.) and triamcinolone (5 mg/kg i.m.) resulted in higher levels of luminescence. (b) Addition of empty AAV5 capsids in a 5:1 ratio (empty to full) with genome containing capsids improved luciferase expression. (c) The percentage of knee joints expressing a positive signal was improved 4 to 9 fold. (d) The clinical score showed a tendency to a decrease in triamcinolone treated animals and a tendency to an initial decrease in liposomal clodronate treated animals.

[0084] FIG. 3: Improvement of intra-articular rAAV5-luciferase expression by addition of empty capsids and/or triamcinolone. In a CIA model, mice (n=13 per group) were injected with 1.5e10 of rAAV5.CMV.Fluc vector (intra-articular) with or without the addition of empty AAV5 capsid (5:1 empty to full) or triamcinolone (5 mg/kg i.m.). Mice were followed weekly for 1 month and thereafter monthly up till 6 months. (a) Arthritis activity was scored at each time point and a clinical score was calculated. Initially arthritis activity was lower in groups treated with triamcinolone. (b) Luminescence increased up till 4 months for all groups, and then remained stable. (c) Generalized estimating equations analysis showed a significant improvement in luminescence when both triamcinolone and empty capsids were added. The clinical score and luminescence are shown per group as averages; error bars, SEM.

[0085] FIG. 4: Comparison of local v.s. systemic triamcinolone administration. In a CIA model (n=18), triamcinolone (5 mg/kg) (or saline) was administered locally (i.a.) or systemically (i.m.), 2 days prior to i.a. administration of rAAV5.CMV.Fluc vector (1.5e10 vg)+empty AAV5 capsid (5:1 empty ratio:full) (intra-articular). Luciferase expression was followed over time.

[0086] FIG. 5: Effect of triamcinolone on spleen size and cell populations in different tissues. Arthritis was induced in several groups (n=5 per group), triamcinolone (5 mg/kg) or saline as control was administered intramuscularly on day 22 (2 days before vector administration). Tissues were analysed by FLOW cytometry 48 hrs after triamcinolone administration. A) the percentage of macrophages (F4/80+, CD68+) in the spleen was similar between saline and triamcinolone treated animals, while percentage of macrophages in the synovium (b) were decreased in triamcinolone group. c-d) spleen weight was significantly reduced in triamcinolone treated animals; the graph and pictures show spleens of groups sacrificed 3 days after triamcinolone/saline administration.

[0087] FIG. 6: Addition of empty capsids in 2 different ratios (5:1 and 20:1) improves intra-articular rAAV5-luciferase expression. Healthy mice (n=7 per group) were injected with rAAV5.CMV.Fluc (1.5e10 vg) intra-articularly, in 2 groups empty AAV5 capsids were added in different ratios. Luciferase expression was measured weekly until mice were sacrificed after 4 weeks. (a) Luminescence at week 4 is shown per group as averages; error bars, SEM. * P<0.05 and ** P<0.01 in groups with empty capsid addition versus the control group that only received genome containing vector (one-tailed unpaired t test).

[0088] FIG. 7: Luciferase expression in air pouch model of synovial inflammation (APSI). rAAV5.CMV.Fluc (3.16e11 vg) was administered into the air pouch of mice (n=5) on d0, d11, or d18 following air pouch formation. In the triamcinolone treated group, triamcinolone was administered (5 mg/kg) by i.m. injection on d9, followed by vector administration on d11. On d30, all mice were sacrificed and air pouch membranes were removed and subjected to luciferase assay. Luciferase expression per group is shown as averages with SEM. Black horizontal line indicates the limit of luciferase detection. The only groups that showed any detectable luciferase expression are when vector was injected on d0, or when triamcinolone was administered 2 days prior to vector.

[0089] FIG. 8: Effect of empty capsid and triamcinolone on intra-articular AAV5 gene expression in healthy mice. Healthy mice (n=17 per group, 34 total injected joints) were injected with rAAV5-CMV-Fluc intra-articularly (1.26e10 vg/joint)+/−empty AAV5 capsid (5:1 empty to full ratio) preceded 2 days prior with i.m. administration of either saline (NaCl) or triamcinolone. Luciferase expression was measured weekly by IVIS up to week 8. a) luciferase measurement over time for all groups. b) Luciferase expression in all groups at week 8. Data show is average per group+SEM. * p<0.05, ** p<0.01, *** p<0.001 as determined by one-tailed Mann Whitney test.

[0090] FIG. 9: Effect of empty capsid and triamcinolone on intra-articular AAV2 gene expression in healthy mice. Healthy mice (n=17 per group, 34 total injected joints) were injected with rAAV2-CMV-Fluc intra-articularly (1.26e10 vg/joint)+/−empty AAV2 capsid (5:1 empty ratio to full) preceded 2 days prior with i.m. administration of either saline (NaCl) or triamcinolone. Luciferase expression was measured weekly by IVIS up to week 4. a) luciferase measurement over time for all groups. b) Luciferase expression in all groups at week 4. Data show is average per group+SEM. ** p<0.01, *** p<0.001 as determined by one-tailed Mann Whitney test.

[0091] FIG. 10: AAV2 vs AA5 capid (VP1) alignment. The sequences show 57% identity as indicated by underscored amino acids.

[0092] The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

EXAMPLES

[0093] Methods

[0094] Vector Production and Empty Capsids

[0095] A rAAV5-transgene vector (Examples 1-7) or rAAV2-trangene vector (Example 8) was produced coding for Firefly Luciferase (Fluc) with a cytomegalovirus (CMV) promoter (rAAV5.CMV.Fluc; Children's Hospital of Philadelphia, Philadelphia, Pa.) as described previously. [Matsushita et al; Gene Ther 1998: 5; 938] In brief, the plasmid encodes the Fluc gene under the control of the CMV promoter and a human growth hormone polyadenylation signal. The transgene cassette is flanked by AAV-2 inverted terminal repeats and is packaged in capsid from AAV5.[Gao G P et al; PNAS 2002; 11854] The genome containing vector and empty AAV capsid particles were purified by combined chromatography and cesium chloride density gradient centrifugation. [Ayuso E, Mingozzi F et al; Gene Ther 2010: 17; 503] Vector titers were determined by qPCR and expressed as vector genomes/ml (vg/ml).

[0096] In Vivo Imaging Experiments in Mice

[0097] Intra-articular rAAV5 expression was investigated in male DBA mice (8-12 week old; Harlan Sprague Dawley, Horst, The Netherlands) in 5 animal experiments. Mice were injected with rAAV5.CMV.Fluc in both knee joints (with or without injection of ankle joints) and monitored periodically for luciferase expression (from 5 days up till 6 months). For animals without arthritis the vector was administered on day 1, in arthritic animals the vector was injected on day 17 or 24 after immunization, at the onset of disease. Animals received between 1.26e10 and 1.5e10 vg per knee joint (in a volume of 5 μl) and 0.75e10 vg per ankle joint (in a volume of 2.5 μl). Empty capsids were co-administered with the genome containing particles in several groups in a 5:1 or 20:1 ratio (ratio expressed as empty capsids to genome containing vectors). Groups consisted of 5 to 18 animals.

[0098] Collagen Induced Arthritis

[0099] Collagen induced arthritis (CIA) was induced by means of an intradermal injection of 100 μl collagen type II (2 mg/ml), diluted 1:1 in CFA (mineral oil and heat-killed M. Tuberculosis 2 mg/ml) (Chondrex Inc., Redmond, Wash., USA). On day 21 a booster injection was administered intraperitoneally containing 100 μg collagen type II dissolved in 100 μl NaCl.

[0100] Arthritis activity was scored 3-weekly from day 18 on with a semi-quantitative scoring system, each mouse paw was analyzed separately (0—normal; 1—swelling and/or erythema of 1 joint; 2—swelling in multiple joints; 3—swelling of all joints; 4—swelling of whole paw and at least one of the following symptoms: ankylosis, loss of function).

[0101] Air Pouch Synovial Inflammation (APSI) Model

[0102] Air pouches were formed as previously described (O'Boyle et al. (2009) FASEB J. 23 (11): 3906-3916). Briefly, 3 mls of air was injected subcutaneously into the back of each animal. The air pouches were kept inflated by re-injection of air as necessary. rAAV5.CMV.Fluc vector (3.16e11 vg) was administered into the pouch in a volume of 1 ml on d0, d11, or d18 post air pouch formation. For triamcinolone treated animals, triamcinolone was administered (5 mg/kg) by i.m. injection 2 days prior to vector administration on d11. On d30, mice were sacrificed and air pouch membrane was removed and snap frozen. Frozen air pouch tissue was homogenized in passive lysis buffer (Promega) and luciferase was measured by standard luciferase assay (Promega).

[0103] Macrophage Inhibition

[0104] Two days prior to vector administration, triamcinolone was administered intra-muscularly (i.m.), similar to the use in RA patients. RA patients receive triamcinolone in a dose of 0.4 to 1.0 mg per kg of bodyweight. Taking into account the faster metabolic rate in mice (factor 12.5), a dose of 5 mg/kg bodyweight was used, administered in a volume of 50 μl. Control groups received an i.m. injection with 50 μl NaCl. In a fourth experiment, i.m. triamcinolone was compared to intra-articular (i.a.) administration two days prior to vector administration, in a comparable dose of (in a volume of 5 μl). A control group received an i.a. injection with 5 μl NaCl.

[0105] Imaging of Luciferase Expression

[0106] Luciferase expression was measured at different time points after vector administration, from day 3 up till 6 months in different experiments. D-luciferin potassium-salt substrate (Caliper Life Sciences, Hopkinton, Mass., USA) was injected intraperitoneally (150 mg/kg of body weight, in a volume of approximately 200 μl). Photon counts were acquired 10 minutes after substrate administration for 5 minutes using a cooled charge-coupled device (CCD) camera system (Photon Imager, Biospace Lab, Paris, France). Light surface images were obtained immediately after each photon counting session to provide an anatomical view of the animal. Image processing and signal intensity quantification and analysis were performed using M3 Vision (Biospace Lab). Images were displayed as a pseudo-color photon count image, superimposed on a gray scale anatomic white-light image, allowing assessment of both bioluminescence intensity and its anatomical source. Regions of interest (ROI) were defined by drawing an elliptical ROI over the knee joint region. The surface area of the ROI was kept constant. The number of photons emitted per second per square centimetre per steradian was calculated as a measure of luciferase activity.

[0107] General Animal Conditions and Ethics Statement

[0108] Immunization, intra-articular injections and in vivo imaging were performed under isoflurane anaesthesia (3% isoflurane and oxygen). At the end of the experiments, animals were sacrificed by cardiac puncture under isoflurane anaesthesia, followed by cervical dislocation, after which hind paws, blood, lymph nodes and spleen were collected. The studies were reviewed and approved by the animal care and use committee of the University of Amsterdam (Amsterdam, The Netherlands; Permit Numbers: ART 102881, ART 102656, ART 102793, ART 102948, and ART 103021) and carried out in strict accordance with the recommendations in the Dutch Law on Animal Welfare (Wet op Dierproeven). Animals were maintained under pathogen-free conditions in the animal facility of the University of Amsterdam.

[0109] FLOW cytometry

[0110] Macrophages in spleen and synovium were analyzed by FLOW cytometry. Briefly, synovial cells were extracted by scraping cells from the joint followed by digestion with Liberase/DNase for 30 min at 37° C. Cells were then washed (PBS/EDTA) and passed through a cell strainer. Synovial cells were centrifuged (1400 rpm, 5 min, 4° C.) and resuspended in FACS buffer (PBS+1% BSA). Due to the low number of cells in the synovium, all animals from each group were pooled. Spleen cells were isolated by mechanical disruption and flushing cells through a cell strainer. Red blood cells were lysed by addition of RBC lysis buffer (Life Technologies), followed by 10 min incubation on ice. Cells were centrifuged and resuspended in FACS buffer. Cells (pooled synovium or splenocytes (1e6 cells) were blocked with 5% normal mouse serum (Sanquin) and stained with F4/80-APC and CD68-FITC labeled antibodies (BD Biosciences). Data was acquired on a BD Canto2 and was analyzed using FlowJo software (FLOWJO LLC, Ashland Oreg.)

[0111] Statistical Analysis

[0112] Luminescence over time was investigated using generalized estimating equations (GEE) to allow for longitudinal analysis (including all available longitudinal data and allowing unequal numbers of repeated measurements) (Twisk (2004) Eur. J. Epidemiol. 19(8):769-776). All other statistics were analyzed using Graphpad Prism (Ja Jolla, Calif., USA). For all tests, differences with a p-value of <0.05 were considered significant.

Example 1

[0113] Inflammation Affects Intra-Articular rAAV5 Transgene Expression

[0114] Fibroblast-like synoviocytes (FLS) are known to increase significantly in the inflamed joint of RA patients (Bartok and Firestein, Immunol Rev, 2010). This is also true for mouse models of RA, including the collagen induced arthritis (CIA) model. As FLS are the primary target cells for AAV5 in the joint, we hypothesized that administration of rAAV5-transgene vector after the onset of inflammation in the CIA model would lead to higher expression, due to a higher number of transduced FLS. To test this hypothesis, we administered an rAAV5-transgene vector encoding the Firefly luciferase gene (rAAV5.CMV.Fluc) intra-articularly in mice with CIA before (d17) or after (d24) the onset of arthritis. Surprisingly, this experiment showed that administration of a rAAV5-transgene vector after the onset of inflammation (day 24) resulted in lower expression per joint as well as a lower percentage of joints expressing the transgene, compared to vector administration before the onset of inflammation (day 17) (FIG. 1).

Example 2

[0115] Immunosuppressive Agents Improve rAAV5 Transgene Expression

[0116] An explanation for decreased expression in animals with inflamed joints could be degradation or neutralization of the vector before it is able to transduce the target cells. During inflammation, there is not only an increase in the number of FLS, but there is also an increase in the number and activation of macrophages, thus we hypothesized that the decreased expression could be due to vector neutralization by macrophages (for example through phagocytosis or opsonization by soluble factors (complement)). To investigate this possibility we studied whether administration of agents that influence macrophage activity/number had an effect on rAAV5-transgene expression. Triamcinolone, a glucocorticosteroid, acts by inhibiting the activation and proliferation of macrophages.[Fauci A S, Dale D C, Balow J E; Ann Intern Med 1976; 84; 304-15] It is a pharmacological agent that is commonly used in humans, for example to treat acute inflammation in the joints of patients with RA. Systemic administration of glucocorticosteroids is also known to exert a local effect by decreasing the number and activity of macrophages in synovial tissue of RA patients. [Gerlag D M et al; Arthritis Rheum 2004; 50(12): 3783] A second agent used to deplete macrophages were clodronate containing liposomes. [van Roijen and Hendrikx, Methods in Molecular Medicine (605) pg 189-203, 2010]. The two agents were administered in separate groups 48 hours before vector administration.

[0117] Both triamcinolone and liposomal clodronate improved rAAV5.CMV.Fluc expression over a period of 4 weeks, showing that either depleting or inhibiting macrophages led to an increase in gene expression (FIG. 2a).

[0118] We hypothesized that another way to avoid macrophage vector neutralization could be to add empty capsid particles upon vector administration. These empty capsids could be acting as a decoy to prevent degradation of the genome containing vector and therefore increasing the chances that full virus particles will be able to reach the target cells. When empty (AAV5) capsids were added to full genome containing capsids in a 5:1 ratio (empty to full), expression improved significantly (FIG. 2b). These data supported our hypothesis that the vector is likely being neutralized by macrophages. In all three groups also an increased percentage of positive joints was seen (FIG. 2c).

[0119] As triamcinolone is an anti-inflammatory agent, arthritis activity was closely monitored. Mice treated with triamcinolone showed a trend towards reduced arthritis activity (FIG. 2d).

Example 3

[0120] Triamcinolone and Decoy Capsids have a Synergistic Effect on rAAV5-Transgene Expression

[0121] We then performed a long term follow up study to assess the duration of expression improved. The study showed that the combination of pharmacological inhibition and empty capsid decoy led to a synergistic enhancement of gene expression. As expected due to its anti-inflammatory effect, arthritis activity was lower in groups treated with triamcinolone up till week 4 (FIG. 3a). Long term arthritis activity was comparable between groups. Luciferase expression was monitored over time for a period of 6 months, remaining stable after an initial increase up to 1 month (FIG. 3b). The effect of addition of triamcinolone and/or empty capsids was analysed longitudinally using generalized estimating equations (GEE), allowing us to include all available longitudinal data and allowing unequal numbers of repeated measurements. A significant increase in luminescence was observed when both compounds were added (ratio of 5.85; p=0.001) (FIG. 3c). Separately the compounds showed a trend towards increased expression (not significant). This shows that the combination of triamcinolone and empty capsid had a synergistic effect on increasing gene expression.

[0122] A similar level of expression was observed in healthy versus arthritic mice (data not shown). Due to technical problems the IVIS data were not available for time point 8 weeks. A total of 15 animals (2-4 per group) was sacrificed prior to the end of the experiment due to reaching a humane endpoint.

[0123] As intra-articular administration of triamcinolone is a standard of care in RA patients, we wanted to determine if the route of triamcinolone administration (systemic vs local) had any effect on efficacy. To investigate this, mice (n=18) were administered triamcinolone locally (intra-articular (i.a.))(or saline as control), or systemically (intra-muscular (i.m.)) 2 days prior to i.a. administration of a composition of rAAV5.CMV.Fluc vector and empty capsids in a ratio of empty capsids:rAAV5.CMV.Fluc vector of 5:1. As can be seen in FIG. 4, pre-treatment of animals with triamcinolone resulted in an enhancement of gene expression. This was true whether the triamcinolone was administered systemically (i.m.) or locally (i.a.), indicating that the route of triamcinolone administration is not a critical factor for efficacy.

Example 4

[0124] Triamcinolone has Differential Effects on Macrophages in Spleen v.s. Synovium

[0125] To further investigate the mechanism of action behind the effect of triamcinolone on transgene expression, an ex vivo analysis was carried out on synovial tissue and splenocytes to assess the effect on the numbers and activity of macrophages and other cell types. Cell populations of the different tissues were compared between triamcinolone and NaCl treated animals by FACS analysis 48 hrs after triamcinolone administration.

[0126] Remarkably, while the relative percentages of macrophages (CD68+, F4-80+) in the spleen remained similar in the triamcinolone treated animals remained (compared with saline) (FIG. 5a), absolute volume of spleen was decreased (4 fold) after triamcinolone treatment (p=0.0011) (FIGS. 5 c and d) at d25, with a reduced difference by day 29. In contrast with the spleen, the percentage of macrophages in the synovium was decreased after triamcinolone treatment (FIG. 5b). Note that given the small numbers of cells that can be extracted from the synovium, we had to pool all of the animals in a group in order to get enough cell counts.

Example 5

[0127] AAV Empty Capsids Improve Transgene Expression in the Absence of Inflammation and Pre-Existing Humoral Immunity

[0128] All previous experiments were all performed in CIA models, in which animals experienced significant inflammation at the joint at the time of vector administration. We then decided to investigate whether the enhancement in luciferase expression could also be seen in healthy joints.

[0129] When empty capsids were added to genome containing particles in 2 different ratios, i.e. in a ratio of empty capsids to rAAV5.CMV.Fluc vector of 5:1 and 20:1, respectively, we observed a dose dependent increase in luminescence (FIG. 6). From day 3 after injection luciferase, expression was increased after addition of empty capsids to genome containing vector in a dose dependent manner (FIG. 6). The increase was 4.8 fold on average in animals injected with empty capsids in a 5:1 ratio to full capsids (p<0.01). A 20:1 ratio (empty to full) improved expression up till 20 fold (P<0.05).

Example 6

[0130] Avoiding/Inhibiting Macrophages Allows for Expression in Air Pouch Synovial Inflammation (APSI) Model

[0131] The air pouch synovial inflammation (APSI) model was initially developed as a way to model human synovium in a mouse. It involves the injection of air under the skin on the back of a mouse. After 6-7 days, a lining membrane will form around this air pouch. This lining is very similar to the synovial lining that forms around the joint cavity, consisting primarily of fibroblast like cells and macrophages. When AAV expressing luciferase was administered into the air pouch on d7 (after formation of air pouch lining), we failed to see any expression, even at high vector doses (data not shown). We hypothesized that perhaps the macrophages lining the air pouch membrane were inhibiting the transduction of the vector (similar to what we have observed in intra-articular injected vector). We tested this hypothesis by either a) administering triamcinolone 2 days prior to vector administration or b) administering vector at d0, prior to the infiltration of macrophages into the air pouch lining. As can be seen in FIG. 7, luciferase expression was only observed when macrophages were either inhibited (e.g., triamcinolone) or were avoided (administration on d0). These data further support the hypothesis that macrophages are detrimental to AAV transduction and that strategies to avoid/inhibit AAV neutralization by macrophages are desired.

Example 7

[0132] Combination of Empty Capsid and Triamcinolone Enhances Intra-Articular AAV5 Gene Expression

[0133] As we have shown that the combination of empty decoy capsid and triamcinolone was effective in increasing gene expression in the inflamed joint of mice with CIA (Example 3), and we have shown that empty capsid alone can increase intra-articular gene expression in healthy joints (Example 5), we wanted to determine if the combination of empty decoy capsid and triamcinolone would enhance gene expression even in the absence of inflammation (healthy joints). To determine this, groups of mice (n=18) were administered triamcinolone (5 mg/kg) or saline (control) by i.m. administration (50 μL total volume). Groups were then administered AAV5-CMV-Fluc (1.26e10 vg/joint) or AAV5-CMV-Fluc+empty AAV5 capsid (empty:full ratio=5:1) by intra-articular injection into both knees (5 μL total volume). Luciferase expression was monitored by live animal IVIS imaging. As can be seen in FIG. 8, both empty decoy capsid and triamcinolone treated animals showed increased gene expression compared with vector alone animals. The combination of empty capsid and triamcinolone gave rise to the highest increase in gene expression levels, similar to what was observed in inflamed (CIA) joints. These data indicate that the combination of triamcinolone and empty capsid are not only effective for increasing expression in diseased joints, but can also increase expression in healthy joints. These results support the hypothesis that the high number of macrophages in the synovial lining (even in healthy joints) are inhibiting AAV mediated expression, and that either adding decoy capsid particles and/or inhibiting macrophage activity can overcome this inhibition, leading to increased gene expression.

Example 8

[0134] Combination of Empty Capsid and Triamcinolone is not Specific for AAV5, but Enhances Intra-Articular Gene Expression from Other Serotypes.

[0135] Our studies thus far have focused on AAV5 as this serotype has excellent tropism for the joint, however we hypothesize that macrophage neutralization of AAV is not serotype specific. This is because AAV update by macrophages is a general phenomenon utilizing scavenger receptors, and thus should not be limited to any one serotype, or any virus type whatsoever as macrophages are known to take up a wide range of viruses and bacteria. To test this hypothesis, we performed an experiment were we evaluated if triamcinolone and empty capsid could enhance gene expression from a serotype that is very different from AAV5, that being AAV2. AAV5 and AAV2 share only 57% homology at the amino acid level (see FIG. 10), making them two of the most diverse serotypes of AAV known. To determine this, an experiment identical to Example 7 was performed, however instead of using AAV5, we used an AAV2 vector. Groups of mice (n=18) were administered triamcinolone (5 mg/kg) or saline (control) by i.m. administration (50 μL total volume). Groups were then administered AAV2-CMV-Fluc (1.26e10 vg/joint) or AAV2-CMV-Fluc+empty AAV2 capsid (empty:full ratio=5:1) by intra-articular injection into both knees (54 total volume). Luciferase expression was monitored by live animal IVIS imaging. As can be seen in FIG. 9, similar to results seen with AAV5, both empty decoy capsid and triamcinolone treated animals showed increased gene expression compared with vector alone animals following AAV2 administration. The combination of empty capsid and triamcinolone gave rise to the highest increase in gene expression levels, similar to what was observed AAV5 treated animals. These data indicate that the combination of triamcinolone and empty capsid are not only effective for increasing expression with AAV5, but is also effective for other diverse serotypes, such as AAV2. Thus, it is clear that that the enhancement of gene expression by macrophage inhibition is not limited to AAV5, but is applicable to all AAV serotypes.

[0136] Given that empty AAV2 and AAV5 capsid were both able to increase gene expression, it follows that this enhancement of expression is not specific for a specific capsid serotype. We therefore conclude that the serotype of the empty capsid need not be of the same serotype as the full genome containing vector and any empty capsid serotype (natural or mutant) should be able to enhance intra-articular expression from any other AAV vector serotype (natural or mutant).