Immunosuppression decreases inflammation and increases AAV6-hSERCA2a-mediated SERCA2a expression

Abstract

The calcium pump SERCA2a (sarcoplasmic reticulum calcium ATPase 2a), which plays a central role in cardiac contraction, shows decreased expression in heart failure (HF). Increasing SERCA2a expression in HF models improves cardiac function. We used direct cardiac delivery of adeno-associated virus encoding human SERCA2a (AAV6-hSERCA2a) in HF and normal canine models to study safety, efficacy, and the effects of immunosuppression. Tachycardic-paced dogs received left ventricle (LV) wall injection of AAV6-hSERCA2a or solvent. Pacing continued postinjection for 2 or 6 weeks, until euthanasia. Tissue/serum samples were analyzed for hSERCA2a expression (Western blot) and immune responses (histology and AAV6-neutralizing antibodies). Nonpaced dogs received AAV6-hSERCA2a and were analyzed at 12 weeks; a parallel cohort received AAV-hSERCA2a and immunosuppression. AAV-mediated cardiac expression of hSERCA2a peaked at 2 weeks and then declined (to ~50%; p<0.03, 6 vs. 2 weeks). LV end diastolic and end systolic diameters decreased in 6-week dogs treated with AAV6-hSERCA2a (p<0.05) whereas LV diameters increased in control dogs. Dogs receiving AAV6-hSERCA2a developed neutralizing antibodies (titer ≥1:120) and cardiac cellular infiltration. Immunosuppression dramatically reduced immune responses (reduced inflammation and neutralizing antibody titers <1:20), and maintained hSERCA2a expression. Thus cardiac injection of AAV6-hSERCA2a promotes local hSERCA2a expression and improves cardiac function. However, the hSERCA2a protein level is reduced by host immune responses. Immunosuppression alleviates immune responses and sustains transgene expression, and may be an important adjuvant for clinical gene therapy trials.

FIG. 1.

Outline of canine toxicology study. (A) Table of study groups. (B) Schematic of the timeline of dog pacing, vector delivery, and immunosuppression. W, weeks.

FIG. 2.

AAV6-mediated human SERCA2a expression in dog heart. Western blots of cardiac extracts of high-dose sites were analyzed with rabbit anti-hSERCA2a antiserum and anti-GAPDH. Human heart (H) and solvent-injected dog heart (C) extracts served as the positive and negative controls, respectively. (A) Western blot images from 2-week tachycardic-paced dogs receiving AAV6-hSERCA2a (lanes 1–5) or solvent (C2); 6-week tachycardic-paced dogs receiving AAV6-hSERCA2a (lanes 6–11) or solvent (C6); 12-week nonpaced dogs receiving AAV6-hSERCA2a (lanes 12–17) or solvent (C12); 12-week nonpaced dogs receiving AAV6-hSERCA2a and immunosuppression (lanes 18–22) or solvent and immunosuppression (C12+I). (B) Quantitative comparison of hSERCA2a expression from digitized X-ray films after normalization to a reference dog (D-70-07). Solid column, 2-week dogs plus AAV6-hSERCA2a (n=5); dark gray column, 6-week dogs plus AAV6-hSERCA2a (n=6); light gray column, 12-week normal dogs plus AAV6-hSERCA2a (n=6); open column, 12-week normal dogs with immunosuppression plus AAV6-hSERCA2a (n=5). IS, received immunosuppression; W, weeks.

FIG. 3.

AAV genome copies at injection site at time of sacrifice. AAV genome copy number per microgram of dog genomic DNA was determined by GLP-level quantitative PCR. Each column represents the mean of high-dose or low-dose sites. (A) Data for high-dose injection sites. (B) Data for low-dose injection sites. IS, received immunosuppression; W, weeks. Two- and 6-week dogs received tachycardic pacing; 12-week dogs did not.

FIG. 4.

Development of neutralizing antibodies (NAbs) against AAV6 after vector injection. (A) Paced dogs with survival period of 2 and 6 weeks after vector administration. (B) Nonpaced dogs with survival period of 12 weeks after vector administration. NAb assays were performed on blood samples collected before vector administration and at weekly or biweekly intervals after vector administration. The serum dilution value is the average of the group. CP, chromatographically purified AAV6-hSERCA2a; GLP, Good Laboratory Practice-grade preparation of AAV6-hSERCA2a purified by CsCl gradient density centrifugation; n, number of dogs per group.

FIG. 5.

Hematoxylin and eosin-stained cardiac tissues from paced dogs receiving AAV6-hSERCA2a treatment for 2 or 6 weeks. (a) Noninjection site of a tachycardic-paced control dog 2 weeks after solvent injection. (b) Solvent injection site of the same dog in (a). (c) Noninjection site of a tachycardic-paced dog 2 weeks after receiving AAV6-hSERCA2a. (d) High-dose injection site of the same dog in (c). (e) Noninjection site of a tachycardic-paced control dog 6 weeks after solvent injection. (f) Solvent injection site of the same dog in (e). (g) Noninjection site of tachycardic-paced dog 6 weeks after receiving AAV6-hSERCA2a. (h) High-dose injection site of the same dog in (g). The scale bar is indicated.

FIG. 6.

(A) Hematoxylin and eosin (H&E)-stained cardiac tissues from nonpaced dogs 12 weeks after receiving AAV6-hSERCA2a. A1: Noninjection site of a control dog. A2: Solvent injection site of the same dog in A1. A3: Noninjection site of a dog receiving AAV6-hSERCA2a. A4: High-dose injection site of the same dog in A3. A5: Noninjection site of an immunosuppressed dog receiving AAV6-hSERCA2a. A6: High-dose site of the same dog in A5. (B) Severity score of fibrosis and chronic inflammation in high dose-injected heart samples. Pathological severity scores, determined by an independent pathology laboratory, are shown for H&E sections of high-dose LV sites from dogs injected with GLP-grade virus. The severity grade for fibrosis and chronic inflammation is described in Materials and Methods. The average severity score is from two high dose-injected sites within one heart and subsequently averaged between two dogs in each group (i.e., data from n=2 per group). Fibrosis in the 6-week control was scored zero. AAV, received AAV6-hSERCA2a; CTL, control (received solvent); IS, received 8 weeks of immunosuppression starting 4 weeks after injections; W, weeks after injections. (C) Immunofluorescently stained cardiac tissues from nonpaced dogs 12 weeks after receiving AAV6-hSERCA2a. C1, C2: Noninjection site of a control dog. C3, C4: AAV6-hSERCA2a-injected sites. Green, phalloidin (sarcomeres); blue, DAPI (nuclei); red, anti-CD4 (C1, C3) or anti-CD8 (C2, C4). The scale bar is indicated.