Patent Application
20080097384
Turning firstly to
A marker band 10 is provided around the foremost body section 6 in order to aid location in the body. The material of the marker band 10 will therefore depend upon the imaging system used.
Use of the catheter described above in a method in accordance with the invention will now be described with additional reference to
Referring initially to
Once the tip 16 of the catheter is in position, saline is pumped into the outer lumen 14 in order to inflate the balloon 8 until it presses against the walls of the hepatic vein 28 which may be seen in
The ejection of the schematically-depicted nucleic acid 30 is shown in
The effect of this pressurised nucleic acid on the liver cells 32 in this area of the liver is to force the nucleic acid 30 through the walls 34 of the liver cells as is shown schematically in
In one example, the therapy is continued in this manner for up to 10 minutes, preferably 1 to 5 minutes and a volume of between 100 ml and a litre is administered depending upon the relative strength of the patient.
Once administration has finished and typically after a further period of 5-20, e.g. 10 mins, the guide wire 4 is replaced down the central lumen 12, the balloon 8 is deflated by withdrawing saline therefrom. This allows blood and some of the introduced liquid to flow to the heart 24. The catheter 2 is then removed by sliding it over the guide wire 4 and finally the guide wire 4 is removed.
Thus in accordance with the described apparatus and methods, an improved method of gene therapy exhibiting significantly higher transfection efficiencies in hepatic liver cells is disclosed.
A further embodiment of the invention is shown in
Use of the catheter 36 shown in
Further embodiments of the invention are shown in
It will be appreciated by those skilled in the art that only certain preferred embodiments of the invention have been described and that there are many variations and modifications possible within the scope of the invention. For example, a centrally guided catheter is not essential and for example a monorail catheter could be used instead. It is also envisaged that the cells undergoing the described therapy may be subjected to ultrasound or other suitable form of radiation in order to enhance the transfection thereof by the nucleic acid. An ultrasonic vibrator e.g. a piezo-electric oscillator could be provided on the catheter for this purpose.
The invention is further described in the following Examples:
The following protocol was performed on 2 pigs of around 40 kg.
The pigs were put under general anaesthetic. A catheter was introduced in the neck vein (external jugular). The catheter had 2 channels; one central channel that can carry an introducer (e.g. a guide wire) and another that can be used to inflate a balloon.
The catheter was pushed down from the neck veins under image intensifier to the superior vena cava, right heart, supra-hepatic vena cava until it reached one of the 3 hepatic veins. For the purpose of this experiment the left hepatic vein is the most suitable.
It was introduced until the catheter did not advance any further.
The balloon was then inflated in order to close completely the lumen of the hepatic vein.
Then the introducer was removed and the nucleic acid injected fast, within a minute or two, under pressure. A volume of 500-1000 ml was injected.
The balloon was kept inflated for about 10 minutes, then deflated slowly and the catheter removed.
The anaesthetic was then discontinued and the animal was recovered. Serial blood tests were performed for 3 weeks to check on any toxicity, liver damage as well as gene expression.
These experiments have shown that this technique was safe. The liver function test remained normal and the animal remained in good health. Significant gene expression was observed.
In this example the plasmid pDERM II expressing rat TPO (thrombopoietin) under the control of a liver specific promoter was injected into the hepatic vein of rats after inferior vena cava (IVC) occlusion and intravenously into the tail vein of rats (controls). 400 g rats were injected with 100 μg of plasmid. The IVC was clamped just above or in the junction with hepatic veins.
TPO is normally produced in the liver and acts on the bone marrow where it stimulates production of platelets by megakaryocytes. The count of platelets (PLT) and white blood cells (WBC) in 1 ml of blood in the systemic circulation were measured in 7 rats and the mean values for each group calculated. The results are shown in Table 1 below, all values are in thousands.
These results show that levels of TPO, i.e. plasmid transfection efficiency, are greater where hydrodynamic injection into the hepatic vein is used.
Patients with Hepatitis C, liver cirrhosis suffer from thrombocytopenia (i.e. low platelet count]. Thrombopoietin (TPO) is secreted from the liver and circulates to the bone marrow and leads to the maturation of megakaryocytes and results in platelet release. Patients with liver cirrhosis have low TPO production and it is proposed to use gene therapy to augment the TPO production in order to bring back the platelet count to normal levels.
Prior to initiating a clinical study we studied the feasibility of this approach in pigs using the hydrodynamic technique of the present invention
Four pigs (median weight 50 kgs) were studied. Prior to gene therapy injection they underwent haematological (full blood count), biochemical (liver function tests, urea and electrolytes as well as serum alpha feto protein measurements) and radiological investigations (ultrasound scan).
Under general anaesthetic and endo-tracheal ventilation a catheter was introduced in the hepatic vein via the internal jugular vein. A contrast material was injected in the catheter after inflation of the balloon in order to verify that the catheter balloon was completely obstructing the hepatic vein and did not allow reflux towards the vena cava.
Three pigs were injected with a plasmid encoding human TPO under the control of a liver specific promoter dissolved in normal saline. This was injected over 20 seconds into the obstructed liver segment. TPO plasmid was injected in a dose of 10 mgs dissolved in 200 mls of normal saline. The fourth pig was injected with a plasmid encoding lac Z which gives blue colouration with beta gal staining.
In each case a single injection was performed. Post-injection blood tests were made in order to assess haematological, biochemical and liver parameters.
As shown in the tables there were no complications associated with this procedure. There were no significant changes in the liver function tests and there was an increase in both platelet count and white blood cells.
Blue colouration in the liver following injection of plasmid lac Z was further evidence of successful transfection.
Plasmid TPO injected according to the method of the invention with doses of 10 mgs and above with a voume in excess of 50 mls can lead to increased serum platelet count and white blood cells. It is proposed that this approach could be used in all forms of liver gene therapy.
In previous pre-clinical models we have shown that it was difficult to increase significantly the TPO levels without the hydrodynamic technique of the present invention. Example 3 shows that our hydrodynamic technique can increase significantly TPO production in a large animal such as pigs (weight over 50 kg).
Therefore a clinical study was initiated in patients with thrombocytopenia to find out whether gene therapy with plasmid TPO injected with the hydrodynamic technique of the present invention can increase the platelet count.
Seven patients (2 males and 5 females), median age 52 yrs were studied. Prior to gene therapy injection they underwent haematological (full blood count), biochemical (liver function tests, urea and electrolytes as well as serum alpha feto protein) and radiological investigations (ultrasound and CT scans).
Following signature of informed consent a catheter was introduced in the hepatic vein via the femoral vein under local anaesthetic. A contrast material was injected in the catheter after inflation of the balloon in order to verify that the catheter balloon is completely obstructing the hepatic vein and does not allow reflux towards the vena cava.
Plasmid TPO dissolved in normal saline was injected for 20 seconds into the obstructed liver segment. The injection was performed by hand, fast and forcefully. TPO plasmid was injected at a dose of 1 mg in patients 1, 2 & 3, in 50 ml, 75 ml and 100 ml respectively. Patient 4 was injected with 2 mg in 150 ml. Patients 5 and 6 were injected with 5 mgs in 150 ml and 200 ml respectively. The seventh patient was injected with 10 mgs in 200 ml and the eighth patient with 10 mg in 250 ml. The balloon was deflated 5 minutes following the injection and the catheter was removed afterwards. Patients were discharged home 2 hours following this procedure. In each case a single injection was performed.
Post-injection blood tests were made in order to assess haematological, biochemical and liver parameters.
There were no complications associated with this procedure. There was no fever or rigors. There was minimal pain in the groin just during the catheter insertion. There were no changes in the liver function tests.
Thus plasmid TPO injected in accordance with the present invention with doses of 5 mg and above and at a volume in excess of 50 ml can lead to increased serum platelet count. This approach potentially could be used in all forms of liver gene therapy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0406728.6 | Mar 2004 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB05/01243 | 3/29/2005 | WO | 00 | 9/28/2007 |
