AAV2-VEGF-B gene therapy failed to induce angiogenesis in ischemic porcine myocardium due to inflammatory responses

Fig. 1. Study protocol. Thirteen animals were followed up until 1-month time point, and seven animals until 6-month time point. PET imaging was performed one day prior to the gene transfer and sacrification. Angiographic measurements and blood sampling were performed at the time of angiographic procedures and sacrification.

Fig. 2. No angiogenesis in AAV2-VEGF-B186 transduced hearts was observed. The mean capillary area was at the same level in the (a1–a2) AAV2-VEGF-B186 hearts compared to (b1–b2) the control group. c1–c2 There was no improvement in myocardial perfusion reserve measured by [15O]H2O-PET. d1–d2 Analysis of [18F]FDG-PET imaging showed no change in FDG uptake at the gene transfer or the infarction area. Results are represented as dot plots separately for each individual animal and as mean ± 95% CI. Scale bar: 100 µm.

Fig. 3. No improvement in cardiac output was seen after 1-month follow-up. Lines represent the group mean ± 95% CI.

Fig. 4. AAV2-vector genome copy numbers remained low in all collected tissues, including the myocardial samples from the gene transfer and infarct areas. AAV2 GT area mean consists of ten independent samples from the target area of each myocardium. Results are expressed as a dot plot, each dot representing an individual animal and as a mean.

Fig. 5. High levels of anti-AAV2 IgG antibodies were detected in the pig sera. Binding anti-AAV2 IgGs were measured from the sera of six animals with a solid-phase ELISA assay before and 1 month after the gene transfers. Each graph represents an individual animal.

Fig. 6. Neutralizing antibodies in the pig sera. nAbs were measured from eight animals before and after the AAV2 gene transfer. Each graph represents an individual animal. The curves present the capability of the serum anti-AAV2 antibodies to prevent AAV2-muSEAP transduction in 293T cells. The framed combined dot plot shows the serum dilution needed to inhibit AAV2-muSEAP transduction efficacy of more than 50%.

Fig. 7. CD3 immunostaining of myocardial sections. Several CD3 positive cells were observed at the gene transfer area of AAV2-transduced animals at 1-month time point, whereas only individual CD3 positive cells were seen in the control samples collected from ischemic animals that had not gone through any gene transfer. Scale bar in 4×: 500 µm. Scale bar in 10×: 100 µm.

Fig. 8. Histology of selected organs and tissues. Hematoxylin–eosin-stained tissues collected from AAV2-transduced animals showed normal histology except in lymph nodes. Both (d1–d2) proximal lymph nodes collected from the thoracic cavity and (e1–e2) distal lymph nodes from the groin showed robust reactivity suggesting a systemic inflammatory response to the AAV2 gene transfer vector. Magnification: ×40 and ×100. a1–a2 lung (b1–b2) kidney (c1–c2) liver, (d1–d2) lymph node proximal (e1–e2) lymph node distal (f1–f2) spleen, (g1–g2) ovario, (h1–h2) brain, (i1–i2) retina.