Development of clinically applicable loco-regional vector delivery strategies

Viktoria Horchemer (Heidelberg)1, M. T. Vo (Heidelberg)1, D. Kehr (Heidelberg)2, E. Meinhardt (Heidelberg)1, M. Pott (Heidelberg)1, P. Raake (Augsburg)3, N. Frey (Heidelberg)4, P. Schlegel (Heidelberg)4

1Universitätsklinikum Heidelberg Innere III, Kardiologie - AG Molekulare Zielstrukturen bei Herzerkrankungen Heidelberg, Deutschland; 2Universitätsklinikum Heidelberg Innere III, Sektion Molekulare und Translationale Kardiologie Heidelberg, Deutschland; 3Universitätsklinikum Augsburg I. Medizinische Klinik Augsburg, Deutschland; 4Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland


Cardiac gene therapy (GT) is a promising treatment option for genetic cardiomyopathies and heart failure. Successful approaches critically depend on (1) a suitable molecular target, (2) a vector with high cardiac tropism and (3) an effective delivery method. Even though current vector technology has generated Adeno-associated viral (AAV) vectors with increased cardiac specificity. Current systemic GT therapies still require extremely high vector doses with dose dependent increase for adverse events. Loco-regional delivery strategies allow a reduction of vector dose while high vector concentration in the target region remain achievable. A critical efficacy factor for these approaches is vector/endothelium contact time. Extending this contact time might improve transduction efficiency.
Since rapid ventricular pacing (RP) reduces net cardiac output and decelerates coronary perfusion as well as microvascular flow, we hypothesized that RP might increase AAV/endothelium contact time and transduction efficiency. 
To develop and evaluate a RP enhanced loco-regional vector delivery method with improved transduction efficiency.
A recombinant AAV6 expressing a firefly Luciferase (rAAV6.HA-fLuc) was used. Pre-screened animals with low neutralizing antibody (nAb, <1:4) titers were randomized into 3 groups to receive 1x1014 viral genome copies (vg)/animal via three different vector delivery protocols: 1) antegrade intracoronary (IC) infusion, 2) microcatheter antegrade IC infusion or 3) rapid pacing-supported microcatheter antegrade IC infusion. 
The rapid pacing delivery protocol included 4 rounds or rapid pacing (30 seconds each) with a two-minute recovery period in between. An external pacemaker was positioned in the right ventricle to induce high rate pacing during vector delivery. Blood samples were collected prior to gene transfer and at pre-specified timepoints, which allowed to closely monitor the safety profile of the gene transfer protocol. 28 days post gene transfer animals were sacrificed, and organs harvested for further molecular analysis. Luciferase activity in different segments of the heart was determined as markers for transduction efficiency. 
Generally Rapid pacing was well tolerated, and no adverse effects were observed. When compared to antegrade gene delivery, microcatheter-supported vector delivery significantly increased myocardia luciferase expression with the highest levels detected in LAD-supplied areas and lower levels in RCA- or LCx-supplied segments. 
Concomitant rapid pacing was safe and able to further increased transgene expression, particularly in the basal LAD segments. Here 6-fold higher luciferase expression was observed compared to animals that underwent antegrade delivery alone. 
RP enhanced AAV vector delivery might be a clinically applicable delivery method which increases loco-regional transduction efficacy in pigs. 
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