Cardiovascular Projects

• Gene Therapy for Arterial Restenosis.
• Gene Therapy for Hypertension.
• Gene Therapy for Pompe's Disease.

Project: Gene Therapy for Arterial Restenosis.

M. Ian Phillips, Ph.D., D.Sc.

Angiotensin II (Ang II) has been implicated as a growth factor in vascular smooth muscle (VSM) and, at least in animal models, Ang II causes the excessive growth of VSM which leads to restenosis in blood vessels after balloon catheterization. The proliferation of vascular muscle after balloon angioplasty causing blood vessels to close again within weeks or months is a serious clinical problem. We propose to approach it by developing antisense DNA to angiotensin II (AT1) receptor mRNA delivered in an adeno-associated virus (AAV) during the balloon angioplasty. We hypothesize that genetic intervention in the blood vessel, after balloon injury, can produce genomic antisense DNA to components of the renin-angiotensin system which will decrease the growth stimulating effects of Ang II and allow full recovery without restenosis.

We have successfully used the rat model for balloon angioplasty and vascular smooth muscle hypertrophy. We have used FITC labelled antisense to AT1 receptors to show that the antisense is taken up into VSM and retained intact in the cells. The vector delivered antisense DNA is taken up by the cells and integrated into the chromosomes. It can then be transcribed and translated to produce antisense mRNA which hybridizes to the AT1-R mRNA and prevents translation. The AAV vector will have a reporter gene for use with atherectomy catheters in humans. The tissue removed by atherectomy will be analyzed for presence of the reporter gene to ensure that the vector had been delivered into the tissue surrounding the area of angioplasty. This approach was tested in rats and will be retested in pigs because of the close similarity between pig vascular biology and human vascular biology. Since the vector is integrated in the chromosome and becomes unnecessary past the critical period for restenosis, the possibility of inserting a "suicide" gene which can be triggered (e.g. by ganciclovir) will be considered. Patients recruited to the GCRC will be cardiology patients, supervised by the Division of Cardiology. Patients will be followed intensively for three months for signs of changes in coronary blood flow and tissue perfusion and thereafter for life, using the resources of the OCI.

References

1. Meng H, Wielbo D, Gyurko R, Phillips MI. Antisense oligonucleotide to AT1 receptor mRNA inhibits central angiotensin induced thirst and vasopressin. Reg Peptides 54:543-551, 1994.
2. Phillips MI, Wielbo D, Gyurko R. Antisense inhibition of hypertension: A new strategy for renin-angiotensin candidate genes. Kid Intern 46:1554-1566, 1994.

Project: Gene Therapy for Hypertension.

M. Ian Phillips, Ph.D., D.Sc.

We are developing antisense DNA in an adeno-associated viral vector (AAV) to integrate angiotensin II (AT1) receptor mRNA or angiotensinogen mRNA antisense in the genome of patients with hypertension. Initially, this will be tested in patients with a familial related angiotensinogen mutant that leads to overexpression of the renin-angiotensin system (RAS) and hypertension. If successful the gene therapy could be extended to a larger population of hypertensives and hypertension in pre-eclampsia. It is the goal of this research to provide, through gene therapy, an accurate, highly specific and effective treatment with few side effects that can overcome a major problem in hypertension treatment, namely the problem of compliance. A single injection, once a year or at longer intervals with effective control of hypertension, would dramatically reduce the need for daily intake of antihypertensive drugs, reducing costs and increasing effectiveness.

We have succeeded in reducing hypertension in spontaneously hypertensive rats (SHR) by the direct application of antisense oligonucleotides to angiotensinogen mRNA and angiotensin type-1 receptor mRNA. The next step is to develop a viral vector to deliver the antisense molecule to the nucleus for integration into the genome in peripheral tissues (e.g. liver) and to test its effectiveness on reducing hypertension. Our preliminary results have accomplished this using an AAV virosome vector in which we have cloned a full-length angiotensinogen and AT1 receptor DNA sequence inserted in the antisense direction. Injection of these vectors directly into liver, kidney and brain in vivo, has so far shown the feasibility of gene transfer by immunocytochemical demonstration of the viral rep gene expression in cells close to the site of injection. In SHR, the antisense to the AT1 receptor in a single injection produced a decrease in blood pressure that lasted for 7-9 days. Therefore, we anticipate that gene therapy with an integrative antisense DNA to AT1-R will produce prolonged decreases in blood pressure due to reduced numbers of AT1 receptors with only a single transfection. Although we have thus far tested only virosomes (liposomes containing Sendai viral coat proteins) we are also preparing to test contentional AAV recombinant virus as delivery vectors in collaboration with the Vector Core staff of the Gene Therapy Center. For this purpose we have developed an AAV with cDNA to angiotensinogen mRNA and AT1 receptor mRNA inserted in the antisense direction and under the control of a CMV promoter. We anticipate that within the next five years we will be prepared for gene therapy protocols in hypertensive patients on the GCRC.

Reference

1. Gyurko R, Wielbo D, Phillips MI. Antisense inhibition of AT1 receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin. Reg Peptides 94:167-174, 1993. 
2. Phillips M.  Prolonged reduction of high blood pressure with an in vivo, nonpathogenic adeno-associated viral vector delivery of AT-R mRNA antisense hypertension. 29:374-380, 1997

Project: Gene Therapy for Pompe's Disease.

Barry Byrne, Ph.D.

Description in development