Collateral Therapeutics, Inc.

The presence of anti-adenoviral serotype 5 (Ad5) antibodies may limit the efficacy of Ad5-mediated gene transfer. We therefore tested the hypothesis that intracoronary delivery of an adenovirus encoding human fibroblast growth factor type 4 (Ad5.FGF4) would improve regional myocardial function in an animal model of ischemia when high antibody levels preexist or after a prior intracoronary dose of Ad5. High anti-Ad5 antibody levels were generated in pigs by subcutaneous immunization with an adenovirus encoding LacZ (Ad5.lacZ). Neutralizing antibody levels increased 648-fold (range, 82- to 2108-fold) above preimmunization levels, and persisted for the duration of the study. Myocardial function during pacing-induced ischemia was improved in the ischemic region (p<0.001) at both 2 and 4 weeks after Ad5.FGF4 administration despite the presence of preexisting antibodies to Ad5. In a second set of experiments, the efficacy of a second intracoronary administration of Ad5 was determined by exposing the animals first to an intracoronary dose of Ad5.lacZ, followed 7 weeks later by the therapeutic dose of Ad5.FGF4. After delivery of Ad5.lacZ, anti-Ad5 antibody levels increased 8-fold (range, 1- to 18-fold), but this prior exposure to Ad5 did not prevent the therapeutic effects after subsequent intracoronary dosing with Ad5.FGF4 (p<0.001). In the porcine Ameroid constrictor model of myocardial ischemia the presence of anti-Ad5 antibodies or prior intracoronary dosing with adenovirus does not prevent the ability of Ad5.FGF4, delivered by intracoronary injection, from normalizing regional myocardial function.

We have previously shown that cardiac-directed expression of adenylycyclase type VI (AC(VI)) increases heart function in transgenic mice, and improves heart function and survival in murine cardiomyopathy. However, a potential problem of crossbreeding paradigms that use lines with two constitutively active transgenes is that results can be obfuscated by interactions between transgenes during growth and development.

To develop a model that could be used subsequently to address this generic problem, transgenic mice with tetracycline (tet)-regulated cardiac-specific expression of AC(VI) were generated. In this transgenic strain, the expression of a tet-controlled transactivator (tTA) was under control of the rat alpha-myosin heavy chain promoter. Expression of the AC(VI) gene was driven by a tet-response element (TRE) and a minimal CMV promoter.

Homogenates of hearts showed no change in AC(VI) protein content during tet suppression (doxycycline), confirming successful suppression of transgene expression. Removal of tet suppression for 10 days was associated with a 10-fold increase in cardiac AC(VI) protein content. A similar increase in mRNA was observed (Northern blot analysis). The estimated half-life of newly synthesized cardiac AC(VI) protein was 2-3 days. Isolated cardiac myocytes from animals that had tet-suppression removed for 10 days showed increased cAMP production in response to forskolin stimulation (Transgene Off: 15+/-6 fmol/microg; Transgene On: 39+/-14 fmol/microg; n=5 each group; P=0.004) and also to isoproterenol stimulation (Transgene Off: 20+/-5 fmol/microg; Transgene On: 31+/-12 fmol/microg; n=5 each group; P=0.035) and hearts isolated from these animals showed marked increased left ventricular peak dP/dt in response to dobutamine stimulation (P=0.009) indicating that inducible cardiac AC(VI) is functionally coupled and recruitable.

We have generated transgenic mice with controlled cardiac-specific expression of AC(VI), provided detailed information regarding the kinetics of transgene expression and suppression and estimated the half-life of cardiac AC(VI) protein to be 2-3 days. Finally, we have shown, for the first time, that controlled cardiac-directed expression of a transgene can increase cardiac myocyte cAMP generation and left ventricular contractile function.

Background —We tested the hypothesis that intracoronary injection of a recombinant adenovirus encoding adenylyl cyclase type VI (AC VI ) would increase cardiac function in pigs. Methods and Results —Left ventricular (LV) dP/dt and cardiac output in response to isoproterenol and NKH477 stimulation were assessed in normal pigs before and 12 days after intracoronary delivery of histamine followed by intracoronary delivery of an adenovirus encoding lacZ (control) or AC VI (1.4×10 12 vp). Animals that had received AC VI gene transfer showed increases in peak LV dP/dt (average increase of 1267±807 mm Hg/s; P =0.0002) and cardiac output (average increase of 39±20 mL · kg −1 · min −1 ; P <0.0001); control animals showed no changes. Increased LV dP/dt was evident 6 days after gene transfer and persisted for at least 57 days. Basal heart rate, blood pressure, and LV dP/dt were unchanged, despite changes in cardiac responsiveness to catecholamine stimulation. Twenty-three hour ECG recordings showed no change in mean heart rate or ectopic beats and no arrhythmias. LV homogenates from animals receiving AC VI gene transfer showed increased AC VI protein content ( P =0.0007) and stimulated cAMP production ( P =0.0006), confirming transgene expression and function; basal LV AC activity was unchanged. Increased cAMP-generating capacity persisted for at least 18 weeks ( P <0.0002). Conclusions —Intracoronary injection of a recombinant adenovirus encoding AC provides enduring increases in cardiac function.