In phase I KAT301 trial, intramyocardial adenovirus-mediated vascular endothelial growth factor -D^ΔNΔC (AdVEGF-D) gene therapy (GT) resulted in a significant improvement in myocardial perfusion reserve and relieved symptoms in refractory angina patients at 1-year follow-up without major safety concerns. We investigated the long-term safety and efficacy of AdVEGF-D GT. 30 patients (24 in VEGF-D group and 6 blinded, randomized controls) were followed for 8.2 years (range 6.3–10.4 years). Patients were interviewed for the current severity of symptoms (Canadian Cardiovascular Society class, CCS) and perceived benefit from GT. Medical records were reviewed to assess the incidence of major cardiovascular adverse event (MACE) and other predefined safety endpoints. MACE occurred in 15 patients in VEGF-D group and in five patients in control group (21.5 vs. 24.9 per 100 patient-years; hazard ratio 0.97; 95% confidence interval 0.36–2.63; P = 0.95). Mortality and new-onset comorbidity were similar between the groups. Angina symptoms (CCS) were less severe compared to baseline in VEGF-D group (1.9 vs. 2.9; P = 0.006) but not in control group (2.2 vs. 2.6; P = 0.414). Our study indicates that intramyocardial AdVEGF-D GT is safe in the long-term. In addition, the relief of symptoms remained significant during the follow-up.

Adenoviral vectors are commonly used in clinical gene therapy. Apart from oncolytic adenoviruses, vector replication is highly undesired as it may pose a safety risk for the treated patient. Thus, careful monitoring for the formation of replication-competent adenoviruses (RCA) during vector manufacturing is required. To render adenoviruses replication deficient, their genomic E1 region is deleted. However, it has been known for a long time that during their propagation, some viruses will regain their replication capability by recombination in production cells, most commonly HEK293. Recently developed RCA assays have revealed that many clinical batches contain more RCA than previously assumed and allowed by regulatory authorities. The clinical significance of the higher RCA content has yet to be thoroughly evaluated. In this review, we summarize the biology of adenovirus vectors, their manufacturing methods, and the origins of RCA formed during HEK293-based vector production. Lastly, we share our experience using minimally RCA-positive serotype 5 adenoviral vectors based on observations from our clinical cardiovascular gene therapy studies.

Background: Despite tremendous therapeutic advancements, a significant proportion of coronary artery disease patients suffer from refractory angina pectoris, that is, quality-of-life-compromising angina that is non-manageable with established pharmacological and interventional treatment options. Adenoviral vascular endothelial growth factor-D^ΔNΔC (AdVEGF-D)-encoding gene therapy (GT) holds promise for the treatment of refractory angina. Methods: ReGenHeart is an investigator-initiated, multicentre, randomised, placebo-controlled and double-blinded phase 2 clinical trial that aims to study the safety and efficacy of intramyocardially administered angiogenic AdVEGF-D GT for refractory angina. Patients will be randomised in a 2:1 ratio and blocks of six to receive either AdVEGF-D or placebo. Primary endpoints are improvements in functional capacity assessed with the 6 min walking test and angina symptoms with Canadian Cardiovascular Society class after 6 month follow-up. Secondary endpoints are improvements in myocardial perfusion assessed with either positron emission tomography or single-photon emission CT after 6 month follow-up and functional capacity and angina symptoms after 12 months. In addition, changes in the quality of life, the use of angina medication and the incidence of major adverse cardiac and cerebrovascular events will be evaluated. Conclusions: The phase 2 ReGenHeart trial will provide knowledge of the safety and efficacy of AdVEGF-D GT to ameliorate symptoms in refractory angina patients, extending and further testing positive results from the preceding phase 1/2a trial.

Biological bypass through induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy (GT) 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 GT production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 10 ¹⁰ 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 (PCR) RCA assay in a preclinical rabbit model ( n = 21). β-galactosidase encoding nonclinical-grade preparation was used as a nonangiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 10 ¹¹ vp. Efficacy of the products was tested by the combination of contrast pulse sequencing 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 PCR 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 GT products may not be as important major safety issue as previously anticipated.

Background: In patients with suspected coronary artery disease (CAD), diffusely impaired hyperaemic myocardial blood flow (MBF) may indicate a multivessel epicardial CAD but also impaired coronary microcirculation. We assessed whether relative hyperaemic MBF—that is, the ratio of the lowest to the highest regional hyperaemic MBF—predicts obstructive CAD in patients with diffusely impaired hyperaemic MBF. Methods: We studied 874 patients who underwent coronary computed tomography angiography and [ ¹⁵ O]H ₂ O myocardial perfusion imaging with positron emission tomography at Kuopio University Hospital, Finland, in 2017–2023. The final analyses included 64 patients who had diffusely impaired hyperaemic MBF and were subsequently referred to invasive coronary angiography (ICA). Results: In ICA, 34/64 (53%) patients had obstructive CAD. Compared to patients with non‐obstructive CAD, those with obstructive CAD had lower relative hyperaemic MBF: 66% (95% CI 62–69%) versus 52% (95% CI 47–57%), respectively ( p < 0.001). When relative hyperaemic MBF was used to predict the presence of obstructive CAD in ICA, the receiver operating characteristic analysis showed an area under the curve of 0.778 (95% CI 0.666–0.891). In binary logistic regression analysis, relative hyperaemic MBF independently predicted obstructive CAD (OR = 0.92 [95% CI 0.86–0.97], p = 0.004). Conclusion: Obstructive CAD is more likely in patients with lower relative hyperaemic MBF. Consequently, relative hyperaemic MBF may have clinical utility in patients with suspected CAD and diffusely impaired hyperaemic MBF.