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The daunting challenges of gene therapy for malignant disease
Editorials The Daunting Challenges of Gene Therapy for Malignant Disease SEE ARTICLE ON PAGE 1359.
Gene therapy has captured the imaginations of biomedical scientists because of its perceived potential to dramatically improve therapies for the most challenging medical problems. What could be more ‘‘natural,’’ after all, than modifying DNA profiles of individuals or their diseased cells so as to render them genetically resistant to pathogenic anomalies? Given this prospect, it is hardly surprising that neoplastic diseases have drawn the attention of gene therapists. These maladies, often inexorably progressive, psychologically and physically debilitating, and fatal, are matched in their perniciousness only by their prevalence; indeed, they constitute a major cause of death in all developed countries. Current treatment approaches to most neoplasias are as crude as their targets are indomitable: patients are carved with sharp instruments, poisoned, or subjected to dangerous radiation. This is not to deprecate the dedication of oncologists, which often reaches heroic proportions; it is only to point out that our armamentarium is weak. Under these circumstances, one can easily understand why physicians, scientists, and patients look upon gene therapy with eager anticipation. The article by Kanai et al. in this issue of HEPATOLOGY explores the use of adenovirus vectors expressing the herpes thymidine kinase (tk) gene under the regulation of the a-fetoprotein (AFP) promoter for conditional ablation of hepatoma cells. In this in vitro model of gene therapy for hepatocellular carcinoma (HCC), the authors report that the DNA construct is highly effective in killing hepatoma cells that express AFP when ganciclovir (GCV) is administered, and, that neighboring, nontransfected cells are efficiently killed by a bystander effect. The experimental design is based upon the fact that AFP, normally expressed only in the fetus, is reactivated in many cases of HCC. Thus, the experiments exploit unique patterns of gene expression in malignant cells to target those cells specifically for chemotherapeutic ablation. The publication of these findings provides a timely opportunity to examine the problems posed by application of such gene therapy
Abbreviations: tk, herpes thymidine kinase gene; AFP, a-fetoprotein; HCC, hepatocellular carcinoma; GCV, ganciclovir. Received February 15, 1996; accepted March 26, 1996. Address reprint requests to: Jon W. Gordon, M.D., Ph.D., Mt. Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2306-0054$3.00/0
methodologies to tumors in vivo. These problems can be summarized as follows: Tumors are metastatic at the time of diagnosis. By the time signs and symptoms appear, most tumors are advanced. In the case of solid tumors, advanced disease is often characterized by the presence of multiple tumor masses, some of which lie quite distant from the primary lesion. Thus, while stereoscopic insertion of retroviral vectors that produce tk is quite efficient at destroying gliomas in the brains of rats,1 such procedures can only be expected to destroy the mass that is treated directly, and not to impair growth of geographically distant metastatic foci. Thus, such treatments for widespread solid tumors would function as a form of ‘‘genetic surgery,’’ reducing tumor mass but not effecting a cure. Where disseminated hematologic malignancies are concerned, the problem is perhaps even more formidable. A related difficulty is that administration of gene vectors and adjuvant drugs may be imperfect. Where solid tumors are concerned, penetration of gene vectors to all cells may be a serious impediment, because cells embedded in large tumor masses are often difficult to access. The problem extends to drugs administered as adjuvants for gene therapy. If these compounds are unable to penetrate to the genetically engineered cells, they will, of course, be ineffective. A potential approach to these obstacles is genetic engineering of enhanced sensitivity to host immune mechanisms. Golumbek et al.2 expressed interleukin4 in renal carcinoma (Renca) cells. Not only were these cells rejected, but the animals were rendered capable of eliminating inocula of non–interleukin-4–producing Renca cells introduced at a distant site. Similarly, injection of interleukin-12–producing fibroblasts into sarcomas of mice dramatically impaired tumor growth, and systemic interleukin-12 administration reduced established lung metastases.3 These findings hold some promise for immune enhancement as a gene therapy approach, though some serious problems may still remain (see below). It has also been proposed4 that, under circumstances where a substantial tumor risk is known to exist, ablation genes might be prophylactically inserted into the organ(s) at risk. An example of this strategy would be insertion of AFP/tk genes into livers of individuals with chronic active hepatitis. If a founder tumor cell possessed the ablation gene prior to neoplastic degeneration, all mitotic descendants of that founder, regardless of their location, would be amenable to ganciclovir ablation. As discussed below, for immunological enhancement, this strategy could still fail; moreover, insertion of genes as a prophylactic measure could cause some ethical concerns.
1696
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WBS: Hepatology
HEPATOLOGY Vol. 23, No. 6, 1996
GORDON
The bystander effect may not always be beneficial. In the report discussed here, a pronounced bystander effect, due presumably to transfer of phosphorylated products of GCV to neighboring, genetically unaltered cells, is described and discussed as a phenomenon that offers the potential to overcome failure to transfer ablation genes into every tumor cell. However, a bystander effect could prove harmful or even dangerous under circumstances where bystander cells were not malignant and were mitotically active. Killing of such normal cells could lead to toxic effects. In the case of HCC, for example, it is not at all clear that all AFP-producing cells are malignant, because these tumors often arise in a setting of recurrent inflammation, cell damage, and liver regeneration. Of some consolation in this particular case are the findings of Macri and Gordon,4 who expressed AFP/tk genes in transgenic mice. In those studies, adult animals showed residual expression of the tk gene in normal liver. When animals were given ganciclovir for the treatment of HCC induced by expression of an albumin/SV40 construct, expression of tk in the normal liver did not appear to enhance toxicity. However, the potential hazards of a bystander effect cannot be ignored on the basis of negative results in a single model system. Conditional ablation could work too well. While gene therapy for cancer could fail because some cells might escape gene transfer, those cells that take up conditional ablation genes would become exquisitely sensitive to GCV therapy. Because these cells would be killed en masse during initial rounds of treatment, significant side effects of tumor lysis could be encountered. In our own work (Macri and Gordon, unpublished data, June 1994) transgenic mice carrying AFP/tk genes and treated for HCC were invariably killed by the treatment if the tumors were advanced at the time therapy was initiated. Hyperemic livers were seen histologically. While the precise reason for the animals’ deaths was not pinpointed, it was not GCV toxicity, because administration of far higher doses of the drug was well tolerated by animals when administered in the early phases of HCC. We may infer from the findings that tumor lysis was in some way responsible for the animals’ demise. Where solid tumors are concerned, massive cell death could lead to hemorrhagic necrosis. In areas such as the central nervous system, such events could be catastrophic. Selection of cancer cells resistant to gene therapy might occur. Like all other tumor therapies, gene therapy exerts selective pressure on the cancer. Somatic events that lead to resistant cells is therefore a possibility. Moolten et al.5 inserted immunoglobulin enhancer/ tk transgenes and were able to eliminate lymphomas with GCV. However, when lymphomas recurred in some animals, the recrudescent malignancies were resistant to GCV on the basis of having lost the tk gene. Similarly, in our own work with HCC,4 tumor nodules
5p0e$$0048
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hepa
1697
were visible in livers of AFP/tk mice after prolonged GCV treatment. Cell selection can be a major obstacle to gene therapy by immunological enhancement, because such treatment would exert strong selection for modification of antigenic profiles of malignant cells. Obviously, we would expect the degree of tumor burden to correlate positively with emergence of resistant cells from therapeutic selection. Gene therapy in humans is a clinical procedure. While it may seem obvious to note that gene therapy is a clinical procedure, this fact is often forgotten during this preliminary, exuberant period of vector design and experiments confined to tissue culture and animals. When gene therapy is applied to humans, however, we must anticipate the occurrence of the same complications as are encountered with all clinical studies: Humans are genetically heterogeneous, they vary in age, and each of them has a unique developmental history and underlying health status. Psychosocial factors that affect compliance with treatment regimens, follow-up evaluation, and other components of treatment protocols will also certainly occur. These variables will correspondingly reduce uniformity of response to treatment. Failure to appreciate this point could lead to naive extrapolations of results obtained from model systems to the human situation. There is no question that gene therapy represents a brilliant, potentially wonderful application of recombinant DNA technology. We cannot help but wax enthusiastic when we read such articles as are published in this issue of HEPATOLOGY. However, the sooner we appreciate the inescapable complicating factors confronting gene therapy strategies for malignant disease, the sooner the risk of stunning disappointment will be replaced by steadfast, methodical refinement of this new, powerful, therapeutic tool. JON W. GORDON, MD, PHD Mt. Sinai School of Medicine New York, NY REFERENCES 1. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 1992;256:15501552. 2. Golumbek PT, Lazenby AJ, Levitsky HI, Jaffee LM, Karasuyama H, Baker M, Pardoll DM. Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4. Science 1991; 254:713-716. 3. Zitvogel L, Tahara H, Robbins PD, Storkus WJ, Clarke MR, Nalesnik MA, Lotze MT. Cancer immunotherapy of established tumors with IL-12. J Immunol 1995;155:1393-1403. 4. Macri P, Gordon JW. Delayed morbidity and mortality of albumin/ SV40 T-antigen transgenic mice after insertion of an alpha-fetoprotein/herpes virus thymidine kinase transgene and treatment with ganciclovir. Hum Gene Ther 1994;5:175-182. 5. Moolten FL, Wells JM, Heyman RA, Evans RM. Lymphoma regression induced by ganciclovir in mice bearing a Herpes thymidine kinase transgene. Hum Gene Ther 1990;1:125-134.
WBS: Hepatology
Gene therapy has captured the imaginations of biomedical scientists because of its perceived potential to dramatically improve therapies for the most challenging medical problems. What could be more ‘‘natural,’’ after all, than modifying DNA profiles of individuals or their diseased cells so as to render them genetically resistant to pathogenic anomalies? Given this prospect, it is hardly surprising that neoplastic diseases have drawn the attention of gene therapists. These maladies, often inexorably progressive, psychologically and physically debilitating, and fatal, are matched in their perniciousness only by their prevalence; indeed, they constitute a major cause of death in all developed countries. Current treatment approaches to most neoplasias are as crude as their targets are indomitable: patients are carved with sharp instruments, poisoned, or subjected to dangerous radiation. This is not to deprecate the dedication of oncologists, which often reaches heroic proportions; it is only to point out that our armamentarium is weak. Under these circumstances, one can easily understand why physicians, scientists, and patients look upon gene therapy with eager anticipation. The article by Kanai et al. in this issue of HEPATOLOGY explores the use of adenovirus vectors expressing the herpes thymidine kinase (tk) gene under the regulation of the a-fetoprotein (AFP) promoter for conditional ablation of hepatoma cells. In this in vitro model of gene therapy for hepatocellular carcinoma (HCC), the authors report that the DNA construct is highly effective in killing hepatoma cells that express AFP when ganciclovir (GCV) is administered, and, that neighboring, nontransfected cells are efficiently killed by a bystander effect. The experimental design is based upon the fact that AFP, normally expressed only in the fetus, is reactivated in many cases of HCC. Thus, the experiments exploit unique patterns of gene expression in malignant cells to target those cells specifically for chemotherapeutic ablation. The publication of these findings provides a timely opportunity to examine the problems posed by application of such gene therapy
Abbreviations: tk, herpes thymidine kinase gene; AFP, a-fetoprotein; HCC, hepatocellular carcinoma; GCV, ganciclovir. Received February 15, 1996; accepted March 26, 1996. Address reprint requests to: Jon W. Gordon, M.D., Ph.D., Mt. Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2306-0054$3.00/0
methodologies to tumors in vivo. These problems can be summarized as follows: Tumors are metastatic at the time of diagnosis. By the time signs and symptoms appear, most tumors are advanced. In the case of solid tumors, advanced disease is often characterized by the presence of multiple tumor masses, some of which lie quite distant from the primary lesion. Thus, while stereoscopic insertion of retroviral vectors that produce tk is quite efficient at destroying gliomas in the brains of rats,1 such procedures can only be expected to destroy the mass that is treated directly, and not to impair growth of geographically distant metastatic foci. Thus, such treatments for widespread solid tumors would function as a form of ‘‘genetic surgery,’’ reducing tumor mass but not effecting a cure. Where disseminated hematologic malignancies are concerned, the problem is perhaps even more formidable. A related difficulty is that administration of gene vectors and adjuvant drugs may be imperfect. Where solid tumors are concerned, penetration of gene vectors to all cells may be a serious impediment, because cells embedded in large tumor masses are often difficult to access. The problem extends to drugs administered as adjuvants for gene therapy. If these compounds are unable to penetrate to the genetically engineered cells, they will, of course, be ineffective. A potential approach to these obstacles is genetic engineering of enhanced sensitivity to host immune mechanisms. Golumbek et al.2 expressed interleukin4 in renal carcinoma (Renca) cells. Not only were these cells rejected, but the animals were rendered capable of eliminating inocula of non–interleukin-4–producing Renca cells introduced at a distant site. Similarly, injection of interleukin-12–producing fibroblasts into sarcomas of mice dramatically impaired tumor growth, and systemic interleukin-12 administration reduced established lung metastases.3 These findings hold some promise for immune enhancement as a gene therapy approach, though some serious problems may still remain (see below). It has also been proposed4 that, under circumstances where a substantial tumor risk is known to exist, ablation genes might be prophylactically inserted into the organ(s) at risk. An example of this strategy would be insertion of AFP/tk genes into livers of individuals with chronic active hepatitis. If a founder tumor cell possessed the ablation gene prior to neoplastic degeneration, all mitotic descendants of that founder, regardless of their location, would be amenable to ganciclovir ablation. As discussed below, for immunological enhancement, this strategy could still fail; moreover, insertion of genes as a prophylactic measure could cause some ethical concerns.
1696
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hepa
WBS: Hepatology
HEPATOLOGY Vol. 23, No. 6, 1996
GORDON
The bystander effect may not always be beneficial. In the report discussed here, a pronounced bystander effect, due presumably to transfer of phosphorylated products of GCV to neighboring, genetically unaltered cells, is described and discussed as a phenomenon that offers the potential to overcome failure to transfer ablation genes into every tumor cell. However, a bystander effect could prove harmful or even dangerous under circumstances where bystander cells were not malignant and were mitotically active. Killing of such normal cells could lead to toxic effects. In the case of HCC, for example, it is not at all clear that all AFP-producing cells are malignant, because these tumors often arise in a setting of recurrent inflammation, cell damage, and liver regeneration. Of some consolation in this particular case are the findings of Macri and Gordon,4 who expressed AFP/tk genes in transgenic mice. In those studies, adult animals showed residual expression of the tk gene in normal liver. When animals were given ganciclovir for the treatment of HCC induced by expression of an albumin/SV40 construct, expression of tk in the normal liver did not appear to enhance toxicity. However, the potential hazards of a bystander effect cannot be ignored on the basis of negative results in a single model system. Conditional ablation could work too well. While gene therapy for cancer could fail because some cells might escape gene transfer, those cells that take up conditional ablation genes would become exquisitely sensitive to GCV therapy. Because these cells would be killed en masse during initial rounds of treatment, significant side effects of tumor lysis could be encountered. In our own work (Macri and Gordon, unpublished data, June 1994) transgenic mice carrying AFP/tk genes and treated for HCC were invariably killed by the treatment if the tumors were advanced at the time therapy was initiated. Hyperemic livers were seen histologically. While the precise reason for the animals’ deaths was not pinpointed, it was not GCV toxicity, because administration of far higher doses of the drug was well tolerated by animals when administered in the early phases of HCC. We may infer from the findings that tumor lysis was in some way responsible for the animals’ demise. Where solid tumors are concerned, massive cell death could lead to hemorrhagic necrosis. In areas such as the central nervous system, such events could be catastrophic. Selection of cancer cells resistant to gene therapy might occur. Like all other tumor therapies, gene therapy exerts selective pressure on the cancer. Somatic events that lead to resistant cells is therefore a possibility. Moolten et al.5 inserted immunoglobulin enhancer/ tk transgenes and were able to eliminate lymphomas with GCV. However, when lymphomas recurred in some animals, the recrudescent malignancies were resistant to GCV on the basis of having lost the tk gene. Similarly, in our own work with HCC,4 tumor nodules
5p0e$$0048
05-17-96 22:01:07
hepa
1697
were visible in livers of AFP/tk mice after prolonged GCV treatment. Cell selection can be a major obstacle to gene therapy by immunological enhancement, because such treatment would exert strong selection for modification of antigenic profiles of malignant cells. Obviously, we would expect the degree of tumor burden to correlate positively with emergence of resistant cells from therapeutic selection. Gene therapy in humans is a clinical procedure. While it may seem obvious to note that gene therapy is a clinical procedure, this fact is often forgotten during this preliminary, exuberant period of vector design and experiments confined to tissue culture and animals. When gene therapy is applied to humans, however, we must anticipate the occurrence of the same complications as are encountered with all clinical studies: Humans are genetically heterogeneous, they vary in age, and each of them has a unique developmental history and underlying health status. Psychosocial factors that affect compliance with treatment regimens, follow-up evaluation, and other components of treatment protocols will also certainly occur. These variables will correspondingly reduce uniformity of response to treatment. Failure to appreciate this point could lead to naive extrapolations of results obtained from model systems to the human situation. There is no question that gene therapy represents a brilliant, potentially wonderful application of recombinant DNA technology. We cannot help but wax enthusiastic when we read such articles as are published in this issue of HEPATOLOGY. However, the sooner we appreciate the inescapable complicating factors confronting gene therapy strategies for malignant disease, the sooner the risk of stunning disappointment will be replaced by steadfast, methodical refinement of this new, powerful, therapeutic tool. JON W. GORDON, MD, PHD Mt. Sinai School of Medicine New York, NY REFERENCES 1. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 1992;256:15501552. 2. Golumbek PT, Lazenby AJ, Levitsky HI, Jaffee LM, Karasuyama H, Baker M, Pardoll DM. Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4. Science 1991; 254:713-716. 3. Zitvogel L, Tahara H, Robbins PD, Storkus WJ, Clarke MR, Nalesnik MA, Lotze MT. Cancer immunotherapy of established tumors with IL-12. J Immunol 1995;155:1393-1403. 4. Macri P, Gordon JW. Delayed morbidity and mortality of albumin/ SV40 T-antigen transgenic mice after insertion of an alpha-fetoprotein/herpes virus thymidine kinase transgene and treatment with ganciclovir. Hum Gene Ther 1994;5:175-182. 5. Moolten FL, Wells JM, Heyman RA, Evans RM. Lymphoma regression induced by ganciclovir in mice bearing a Herpes thymidine kinase transgene. Hum Gene Ther 1990;1:125-134.
WBS: Hepatology