Componente Humano

Adenosine is regularly used to vasodilate the coronary circulation in the clinical setting, a process which could theoretically increase the possibility of adenoviral transfer. To test this hypothesis we performed a number of experiments delivering 1mg of adenosine (Sigma) into the coronary vasculature between 3 and 15 minutes before delivery of adenovirus.

Recently experiments have been carried out using Langendorff perfusion to assess gene delivery with rAdS. To increase the efficiency of gene transfer these investigators infused recombinant adenovirus in nominally calcium-free buffer, to increase the capillary permeability. These investigators demonstrated an increase in gene transfer efficiency, but not wet weight of the heart, a measure of vascular permeability. Therefore to investigate whether using a different vehicle to deliver the recombinant adenovirus would enhance delivery in vivo we performed additional experiments using nominally calcium-free solution. Recombinant adenovirus at 1 x 10^^ pfu/ml (in 0.1ml virus storage medium) was added to 0.9ml of Krebs-Henseleit solution with 50pM free calcium and delivered over 1 minute into the circumflex coronary artery.

2.4 Occlusive Coronary Engagement

A further impediment to intracoronary gene delivery in vivo is that virus is only in the coronary circulation for a very short period of time, and that it is diluted with blood (Donahue et al., 1997). Although the dilution of blood can be overcome by occluding the coronary artery, which would also increase the time the virus is in the coronary circulation, this results in transient ischaemia, as can be observed by ST elevation of the ECG. This can easily lead to VF, which is recalcitrant to DC cardioversion in the rabbit, with the chest closed. To perform occlusive coronary engagements and to decrease the chance of VF secondary to

ischaemia of the left ventricle, we delivered recombinant adenovirus with a perfluorocarbon emulsion (Chapter 2, Section 1.6.3).

Perfluorocarbons (PFCs) are molecules comprised of carbon and fluorine atoms and are chemically inert and resistant to thermal and radiation damage (Millard, 1994). They have been considered for a number of medical applications as they act as solvents for all common gases, including oxygen and carbon dioxide. They were first demonstrated to support liquid respiration thirty years ago (Clark and Goland, 1966) with mice surviving for several weeks following complete immersion in oxygen saturated FX-80, for one hour. These were subsequently approved in 1990 by the US FDA for adjuvant therapy in coronary angioplasty. The solubility of oxygen at atmospheric pressure in perfluorodecalin is 40.3 ml of oxygen in 100ml of perfluorodecalin (Millard, 1994). Unlike haemoglobin the oxygen solubility of perfluorocarbons is linearly related to the partial pressure of oxygen, and is not saturable.

Unfortunately perfluorocarbons are insoluble in liquids and have to be emulsified before they can be used in vivo, usually in phospholipids (Millard, 1994; Faithfull, 1994). Although the use of perfluorocarbons, for their oxygen carrying capability, is relatively uncommon, Fluosol DA, one of the most commonly used perfluorocarbon emulsions (Reiss, 1994) can dissolve about three times as much oxygen as can normal whole blood. Fluosol DA consists of perfluorodecalin, perfluorotripropylamine, pluronic F-68, and egg phospholipids.

The perfluorocarbon that we used in these studies is based upon perfluorodecalin as detailed in chapter 2, section 1.6.3. Oxygenated perfluorodecalin was mixed with either 0.1 ml of virus storage medium or 0.1ml of rAdS-Rgal at 1 x 10^^ pfu/ml. The rAd5-Rgal was delivered during an occlusive engagement in the circumflex territory, until there was sustained ST elevation, which empirically appeared to be close to that leading to VF, with frequent ventricular ectopy.

2.5 High Pressure Gene Delivery

High-pressure adenoviral gene delivery was achieved using a modified approach of Hajjar et al (1998). The right common carotid artery was cannulated

Chapter 5 Intracoronary Delivery of Recombinant Adenovirus

as before, and a 0.014 high fidelity pressure wire was manipulated into the left ventricle. The pressure wire was connected to an amplifier (RADI Medical Systems PGA10, Uppsala, Sweden) and left ventricular pressure was recorded using the MacLab system (Figure 2-2). This allowed simultaneous measurement of both arterial and left ventricular pressure. A median sternotomy was performed using sterile technique and care taken to avoid the pleura and the pericardium was incised. The left ventricle was entered and a 23G needle was guided to the left ventricular outflow tract under fluoroscopic guidance. 1 x 10^° pfu of rAd5- Rgal in 3mls of saline or nominal calcium-free Krebs-Henseleit buffer was injected whilst the aorta and pulmonary artery were clamped for 10 seconds. Occlusion of the aorta was detected by a rise in intraventricular pressure, dilation of the heart and a drop in systemic arterial pressure. The animal was monitored for 5 minutes prior to closure of the chest and neck, and lOOmg of benzylpenicillin was administered iv.

Figure 2-2 E C G an d Pressure Traces Before and During H igh-Pressure G ene Delivery. P a n e l A shows the E C G and arterial/ ventricular pressure with the high frequency pressure wire (RADI). A t the left h a lf o f the trace the R A D I wire is in the systemic circulation, at the aortic root, when the ventricle is entered the trace changes, with en d diastolic pressures o f app ro xim ately W m m H g a n d E C G changes due to ventricular ectopics resulting from touching the inner surface o f the ventricle with the pressure wire. In pan el B, the onset o f clamping o f the aorta a n d p u lm o n a ry a rte ry is a c c o m p a n ie d by a rise in systolic p re s s u re to approxim ately 2 0 0 m m H g and diastolic pressure to 50m m H g, additionally the E C G Q R S complexes change in morphology.

Figure 2-2 ECG and Pressure Traces Before and During High-Pressure Gene Delivery 0.6 0.4 0.2 -0.2 -0.4

Left Ventricle

Aortic Root

Left Ventricle

Chapter 5 Intracoronary Delivery of Recombinant Adenovirus

3 Results

3.1 Intracoronary Gene Delivery with Recombinant Adenovirus,