- Email: [email protected]
Gene therapy: into the future of surgery
THE LANCET
Gene therapy: into the future of surgery
Simon J Hollingsworth, Stephen G E Barker Surgery tends to be evolutionary, but occasionally events allow it to be revolutionary, as, for example, with the introduction of antiseptics and antibiotics and more recently, safe general anaesthesia. As modern-day practice advances and becomes increasingly specialised, the trend is to become more conservative and minimally invasive. Is it possible that "surgery" could in fact become non-invasive? With an exponential growth in the understanding of the molecular basis of disease-free and disease states, and the ever-increasing means of manipulation and control of these processes, the surgeon's request, "scalpel please" might be replaced by "pass the correct plasmid please".
Principles of gene therapy Molecular "treatment" requires, first, the molecular manipulation or alteration of a designated "target" population of cells. Essentially, this change can be effected in one of two ways--by ex-vivo modification, with the material being re-introduced into the body after modification, or by in-vivo modification, done in situ. Whichever approach is adopted, the treatment will probably involve one of the following: • Gene replacement, which consists of the substitution of a non-active or defective gene by a "new" (or additional), functional copy of the gene, to restore the production of a required protein. This technique is used in, for example, the treatment of adenosine deaminase deficiency or cystic fibrosis, or in the treatment of cancer, for which a wild-type p53 gene may be used to correct mutant p53 expression and so restore normal tumoursuppressor function. • Gene addition, which is the insertion into the cell of a new gene, to enable the production of a protein not normally expressed by that cell. For example, a foreign H L A molecule, co-stimulatory protein, or cytokine may be inserted to enhance or stimulate an immune response to cancer cells, or a "pro-drug" convertase gene (also known as a suicide gene) may be inserted to convert a non-active pro-drug into an a c t i v e metabolite, so rendering the target cells susceptible to drug treatment. e Gene control, which is the alteration or control of expression of a gene. For example, the expression of a mutated oncogene or tumour-suppressor gene in tumour cells might be suppressed by use of antisense molecules to target the RNA and so prevent specific protein production. Applications of gene therapy Gene therapy was first used in 1990, for adenosine deaminase deficiency? Since then, more than 100 clinical gene-therapy trials have been initiated world wide. Although most of the trials have been for the treatment of Lancet 1999; 353 (suppl I): 1 9 - 2 0 Academic Vascular Unit, Department of Surgery, Royal Free and UniversityCollege LondonMedical School, MiddlesexHospital, LondonWIN 8AA, UK (S J Hollingsworth PhD,S G E Barker FRCS) Correspondenceto: Dr S J Hollingsworth Surgery • Voi 353 • April • 1999
;ene delivery to arteR Endoluminal Transfer of genes to inner arterial wall
Gene-transfer solution passed along the catheter.
/outer arterial wal
tumours (predominantly malignant melanoma and haematological disorders), there have also been trials of gene therapies for genetic disorders, AIDS, and cardiovascular disease) For the surgeon, perhaps the most relevant of the gene therapies so far are those offering new approaches to the management of cardiovascular disease and "solid" cancers.
Gene therapy for vascular disease One of the first gene-therapy trials for cardiovascular disease was done in non-diabetic patients with rest pain and ischaemic ulceration of the lower limbs? Although designed primarily to assess the safety of (vascular) gene therapy in human beings, the study examined also the effect of transfer of the gene for vascular endothelial growth factor (VEGF) in induction of angiogenesis and, hence, of improvement of tissue microcirculation in critical limb ischaemia. Patients received escalating doses of V E G F DNA, which was delivered to the luminal endothelium of the profunda femoris artery by use of an angioplasty balloon coated with a hydrogel polymer containing a V E G F plasmid D N A vector. The preliminary findings were of a promising clinical response (increase in blood flow and alleviation of symptoms) in three of seven patients. In another study, Baumgartner and colleagues used a method of direct injection of a V E G F plasmid into the leg muscles of patients with ischaemic limbs, non-healing ulcers, and rest pain? The treatment prompted a significant improvement in indices of ankle-brachial pressure, and contrast angiography showed collateral blood-vessel formation in seven of ten s~19
THE LANCET
limbs. Furthermore, the ischaemic ulcers either healed or improved substantially in four of seven limbs, and successful limb salvage proved possible in three patients who had been advised below-knee amputation. A factor contributing significantly to failure of arterial bypass grafts and to re-stenosis after angioplasty is hyperplasia of intimal smooth-muscle cell (SMCs). When delivered (at the time of surgery) to those sites where SMC intimal hyperplasia causes most difficulty, genes for VEGF or inducible nitric oxide synthase, which can inhibit this cellular proliferation, have shown some efficacy in preventing hyperplasia?,6 The chosen gene may be transferred to the arterial wall from the lumen by use of a balloon catheter (figure, upper), or from the outside of the artery by use of a perivascular collar, so that the artery is bathed with a solution containing the DNA to be transferred (figure, lower). An alternative strategy is to transfer a fusion-gene to the arterial wall, to enable the expression of a protein that acts to bind drugs which, when modified to contain a counter-receptor and given systemically, are then selectively anchored at the site chosen (Yla-Herttuala S, personal communication). Clinical trials of these novel strategies will start soon, with results expected in the next 2-3 years.
Gene therapy for cancer Undoubtedly, cancer remains the area of greatest activity for gene therapy, 7 Strategies for gene therapy of solid tumours fall into four major categories: e Immune gene therapy, by which expression of a cytokine (eg, interleukin-2), a co-stimulatory molecule (eg, B7.1), or a tumour antigen (eg, carcinogenic embyonic antigen) can be used to induce or augment tumour immunity. e Therapy with enzyme or pro-drug systems (eg, HSV-tk/ganciclovir or CD/5-fluorocytosine), whereby target cells are rendered selectively susceptible to a particular drug therapy to help induce immunity • Replacement therapy with turnout-suppression gene, in which the aim is to restore normal tumour-suppressor function (eg, p53). e Antisense therapy, which is used to combat the expression of genes whose mutation, abberant expression, or overexpression has rendered ceils tumorigenic. An example of the clinical use of one of these approaches is the injection of wild-type p53 plasmid
sI20
DNA, which is being investigated in trials of the treatment of colorectal metastases in the liver8 and of primary hepatocellular carcinoma2
Prospects for gene therapy The main issues facing the clinical application of gene therapy are perhaps improvements in the delivery and transfer of genes to the appropriate site, and in control of the expression of the gene that has been transferred. Although cDNA complexed to liposomes, and retroviral, pseudotyped retroviral, and adenoviral vectors are being used to promote gene transfer, gene-delivery systems are at an embryonic stage of their development. That said, gene therapy is becoming a clinical reality. In the short to medium term, gene therapy will most probably serve as an adjuvant to conventional therapies--for example, to prevent spread of metastases after tumour de-bulking. Later, as knowledge of the rholecular basis of disease accumulates, and as genetic screening programmes, themselves a product of molecular medicine, are increasingly introduced, it might be possible to target individuals at risk, for vaccination against the disease, or for early diagnosis and treatment.
References 1
2 3
4
5
6 7 8
9
Blaese RM, Culver KW, Miller AD, et al. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science 1995; 270: 475-80. Marshall E. Gene therapy's growing pains. Science 1995; 269: 1050-55. Isner JM, Walsh K, Symes J, et al. Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease. Circulation 1995; 91: 2687-92. Baumgartner I, Pieczek A, M a n o r O, et al. Constitutive expression of p h V E G F 165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb isehemia [see comments]. Circulation 1998; 9 7 : 1 1 1 4 - 2 3 . Shears L L 2rid, Kibbe MR, M u r d o c k AD, et al. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J A m Coll Surg 1998; 187: 295-306. Yla-Herttuala S. Gene therapy for cardiovascular diseases. Ann Med 1996; 28: 89-93. Roth JA, Cristiano RJ. Gene therapy for cancer: what have we done and where are we going? J Natl Cancer Inst 1997; 8 9 : 2 1 39. Habib NA, Ding SF, el Masry R, et al. Contrasting effects of direct p53 D N A injection in primary and secondary liver tumours. Tumour Targ 1995; 1: 295-98. Habib NA, Ding SF, el Masry R, et al. Preliminary report: the shortterm effects of direct p53 D N A injection in primary hepatocellular carcinomas. Cancer Detect Prey 1996; 20: 103-07.
Surgery • Vol 353 ° April ° 1999
Gene therapy: into the future of surgery
Simon J Hollingsworth, Stephen G E Barker Surgery tends to be evolutionary, but occasionally events allow it to be revolutionary, as, for example, with the introduction of antiseptics and antibiotics and more recently, safe general anaesthesia. As modern-day practice advances and becomes increasingly specialised, the trend is to become more conservative and minimally invasive. Is it possible that "surgery" could in fact become non-invasive? With an exponential growth in the understanding of the molecular basis of disease-free and disease states, and the ever-increasing means of manipulation and control of these processes, the surgeon's request, "scalpel please" might be replaced by "pass the correct plasmid please".
Principles of gene therapy Molecular "treatment" requires, first, the molecular manipulation or alteration of a designated "target" population of cells. Essentially, this change can be effected in one of two ways--by ex-vivo modification, with the material being re-introduced into the body after modification, or by in-vivo modification, done in situ. Whichever approach is adopted, the treatment will probably involve one of the following: • Gene replacement, which consists of the substitution of a non-active or defective gene by a "new" (or additional), functional copy of the gene, to restore the production of a required protein. This technique is used in, for example, the treatment of adenosine deaminase deficiency or cystic fibrosis, or in the treatment of cancer, for which a wild-type p53 gene may be used to correct mutant p53 expression and so restore normal tumoursuppressor function. • Gene addition, which is the insertion into the cell of a new gene, to enable the production of a protein not normally expressed by that cell. For example, a foreign H L A molecule, co-stimulatory protein, or cytokine may be inserted to enhance or stimulate an immune response to cancer cells, or a "pro-drug" convertase gene (also known as a suicide gene) may be inserted to convert a non-active pro-drug into an a c t i v e metabolite, so rendering the target cells susceptible to drug treatment. e Gene control, which is the alteration or control of expression of a gene. For example, the expression of a mutated oncogene or tumour-suppressor gene in tumour cells might be suppressed by use of antisense molecules to target the RNA and so prevent specific protein production. Applications of gene therapy Gene therapy was first used in 1990, for adenosine deaminase deficiency? Since then, more than 100 clinical gene-therapy trials have been initiated world wide. Although most of the trials have been for the treatment of Lancet 1999; 353 (suppl I): 1 9 - 2 0 Academic Vascular Unit, Department of Surgery, Royal Free and UniversityCollege LondonMedical School, MiddlesexHospital, LondonWIN 8AA, UK (S J Hollingsworth PhD,S G E Barker FRCS) Correspondenceto: Dr S J Hollingsworth Surgery • Voi 353 • April • 1999
;ene delivery to arteR Endoluminal Transfer of genes to inner arterial wall
Gene-transfer solution passed along the catheter.
/outer arterial wal
tumours (predominantly malignant melanoma and haematological disorders), there have also been trials of gene therapies for genetic disorders, AIDS, and cardiovascular disease) For the surgeon, perhaps the most relevant of the gene therapies so far are those offering new approaches to the management of cardiovascular disease and "solid" cancers.
Gene therapy for vascular disease One of the first gene-therapy trials for cardiovascular disease was done in non-diabetic patients with rest pain and ischaemic ulceration of the lower limbs? Although designed primarily to assess the safety of (vascular) gene therapy in human beings, the study examined also the effect of transfer of the gene for vascular endothelial growth factor (VEGF) in induction of angiogenesis and, hence, of improvement of tissue microcirculation in critical limb ischaemia. Patients received escalating doses of V E G F DNA, which was delivered to the luminal endothelium of the profunda femoris artery by use of an angioplasty balloon coated with a hydrogel polymer containing a V E G F plasmid D N A vector. The preliminary findings were of a promising clinical response (increase in blood flow and alleviation of symptoms) in three of seven patients. In another study, Baumgartner and colleagues used a method of direct injection of a V E G F plasmid into the leg muscles of patients with ischaemic limbs, non-healing ulcers, and rest pain? The treatment prompted a significant improvement in indices of ankle-brachial pressure, and contrast angiography showed collateral blood-vessel formation in seven of ten s~19
THE LANCET
limbs. Furthermore, the ischaemic ulcers either healed or improved substantially in four of seven limbs, and successful limb salvage proved possible in three patients who had been advised below-knee amputation. A factor contributing significantly to failure of arterial bypass grafts and to re-stenosis after angioplasty is hyperplasia of intimal smooth-muscle cell (SMCs). When delivered (at the time of surgery) to those sites where SMC intimal hyperplasia causes most difficulty, genes for VEGF or inducible nitric oxide synthase, which can inhibit this cellular proliferation, have shown some efficacy in preventing hyperplasia?,6 The chosen gene may be transferred to the arterial wall from the lumen by use of a balloon catheter (figure, upper), or from the outside of the artery by use of a perivascular collar, so that the artery is bathed with a solution containing the DNA to be transferred (figure, lower). An alternative strategy is to transfer a fusion-gene to the arterial wall, to enable the expression of a protein that acts to bind drugs which, when modified to contain a counter-receptor and given systemically, are then selectively anchored at the site chosen (Yla-Herttuala S, personal communication). Clinical trials of these novel strategies will start soon, with results expected in the next 2-3 years.
Gene therapy for cancer Undoubtedly, cancer remains the area of greatest activity for gene therapy, 7 Strategies for gene therapy of solid tumours fall into four major categories: e Immune gene therapy, by which expression of a cytokine (eg, interleukin-2), a co-stimulatory molecule (eg, B7.1), or a tumour antigen (eg, carcinogenic embyonic antigen) can be used to induce or augment tumour immunity. e Therapy with enzyme or pro-drug systems (eg, HSV-tk/ganciclovir or CD/5-fluorocytosine), whereby target cells are rendered selectively susceptible to a particular drug therapy to help induce immunity • Replacement therapy with turnout-suppression gene, in which the aim is to restore normal tumour-suppressor function (eg, p53). e Antisense therapy, which is used to combat the expression of genes whose mutation, abberant expression, or overexpression has rendered ceils tumorigenic. An example of the clinical use of one of these approaches is the injection of wild-type p53 plasmid
sI20
DNA, which is being investigated in trials of the treatment of colorectal metastases in the liver8 and of primary hepatocellular carcinoma2
Prospects for gene therapy The main issues facing the clinical application of gene therapy are perhaps improvements in the delivery and transfer of genes to the appropriate site, and in control of the expression of the gene that has been transferred. Although cDNA complexed to liposomes, and retroviral, pseudotyped retroviral, and adenoviral vectors are being used to promote gene transfer, gene-delivery systems are at an embryonic stage of their development. That said, gene therapy is becoming a clinical reality. In the short to medium term, gene therapy will most probably serve as an adjuvant to conventional therapies--for example, to prevent spread of metastases after tumour de-bulking. Later, as knowledge of the rholecular basis of disease accumulates, and as genetic screening programmes, themselves a product of molecular medicine, are increasingly introduced, it might be possible to target individuals at risk, for vaccination against the disease, or for early diagnosis and treatment.
References 1
2 3
4
5
6 7 8
9
Blaese RM, Culver KW, Miller AD, et al. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science 1995; 270: 475-80. Marshall E. Gene therapy's growing pains. Science 1995; 269: 1050-55. Isner JM, Walsh K, Symes J, et al. Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease. Circulation 1995; 91: 2687-92. Baumgartner I, Pieczek A, M a n o r O, et al. Constitutive expression of p h V E G F 165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb isehemia [see comments]. Circulation 1998; 9 7 : 1 1 1 4 - 2 3 . Shears L L 2rid, Kibbe MR, M u r d o c k AD, et al. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J A m Coll Surg 1998; 187: 295-306. Yla-Herttuala S. Gene therapy for cardiovascular diseases. Ann Med 1996; 28: 89-93. Roth JA, Cristiano RJ. Gene therapy for cancer: what have we done and where are we going? J Natl Cancer Inst 1997; 8 9 : 2 1 39. Habib NA, Ding SF, el Masry R, et al. Contrasting effects of direct p53 D N A injection in primary and secondary liver tumours. Tumour Targ 1995; 1: 295-98. Habib NA, Ding SF, el Masry R, et al. Preliminary report: the shortterm effects of direct p53 D N A injection in primary hepatocellular carcinomas. Cancer Detect Prey 1996; 20: 103-07.
Surgery • Vol 353 ° April ° 1999