Efficacy and Safety of Clinical Grade hVEGF-DΔNΔC Gene Therapy Containing Residual Replication-Competent Adenoviruses.

Biological bypass via induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy is a new concept for the treatment of cardiac ischemia. Serotype 5 adenoviruses are used in the clinical trials for transferring the VEGF-D cDNA into the ischemic myocardium. However, the presence of replication-competent vectors in the adenovirus products is a widely recognized problem that may pose a potential safety risk to the treated patients. We compared three different VEGF-D gene therapy production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 1010 viral particles (vp): < 10 RCA (VEGF-D L-RCA1), 10 - 100 RCA (VEGF-D H-RCA2), and 100 - 200 RCA (VEGF-D H-RCA3), as measured by a novel droplet digital polymerase chain reaction (ddPCR) RCA assay in a preclinical rabbit model (n = 21). β-galactosidase encoding non-clinical grade preparation (Adβ-Gal) was used as a non-angiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 1011 vp. Efficacy of the products was tested by the combination of contrast pulse sequencing (CPS) ultrasound and modified Miles assay as well as quantifying the total cross-sectional area of capillaries. Safety, immunogenicity, toxicity, biodistribution and shedding were assessed by general histology, serial measurements of C-reactive protein, white blood-cell count and body temperature as well as using quantitative real time polymerase chain reaction (qPCR) with primers targeted to the VEGF-D and replication-permitting E1 sequences. We found no significant differences in the efficacy or safety between the study groups. Most importantly, no detectable presence of RCA-specific E1 sequence was found in any samples tested, indicating that no detectable vector replication took place in vivo. We conclude that relatively low levels of RCA in adenoviral gene therapy products may not be as important major safety issue as previously anticipated.