- Email: [email protected]
Suicide genes: past, present and future perspectives
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53 Burlingham, W.J. (1996) Chimerism after organ transplantation: is there any clinical significance? Clin. Transplant. 10, 110–117 54 Kimikawa, M. et al. (1997) Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys. Transplantation 64, 709–716 55 Greenstein, J.L. and Sachs, D.H. (1997) The use of tolerance for transplantation across xenogeneic barriers. Nat. Biotechnol. 15, 235–238 56 Sablinski, T. et al. (1997) Pig to monkey bone marrow and kidney xenotransplantation. Surgery 121, 381–391 57 Naji, A. (1996) Induction of tolerance by intrathymic inoculation of alloantigen. Curr. Opin. Immunol. 8, 704–709
58 Allen, M.D. et al. (1997) Prolonged allogeneic and xenogeneic microchimerism in unmatched primates without immunosuppression by intrathymic implantation of CD341 donor marrow cells. Cell. Immunol. 181, 127–138 59 Groth, C.G. et al. (1994) Transplantation of porcine fetal pancreas to diabetic patients. Lancet 344, 1402–1404 60 Paradis, K. et al. (1999) Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 285, 1236–1241 61 Patience, C. et al. (1997) Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3, 282–286
Suicide genes: past, present and future perspectives Shangara Lal, Ulrich M. Lauer, Dietrich Niethammer, James F. Beck and Paul G. Schlegel Disappointing morbidity and mortality rates in many disorders
‘I
f one considers the purpose of a promoter/enhancer sequence for sustained have necessitated the development of drug to be to restore normal funcand optimal gene expression; and (5) a means several ‘biological’ therapies. One tion of some particular process in for controlling the level of gene expression. approach involves the use of suicide the body, then DNA would be Suicide gene therapy is one such strategy considered to be the ultimate drug…’ The that uses the principle of inserting a sogenes. Here, Shangara Lal and foundations of molecular medicine rest called ‘suicide gene’ into the target genome. colleagues discuss the development squarely on Watson and Crick’s discovery of This gene encodes for an enzyme that conof this branch of gene therapy in cell the structure and function of DNA. This verts an inactive prodrug into its cytotoxic and the subsequent discovery of mRNA has metabolite(s) so that, upon introduction of lines and animal models, and led to the formulation of the ‘central dogma’ the prodrug, the target cell effectively is examine current studies, therapeutic that dictates the flow of genetic information compelled to commit suicide. applications and clinical trials. from DNA to RNA to protein. Aberrations along this pathway can manifest as clinical Evolving methods of gene delivery disorders. Morbidity and mortality rates for a variety of malignant cancers, autoimmune and metabolic disorders Several techniques have been developed for transferring genes have only marginally improved over the past 15 or so years and care across the cell membrane (Table 1), many of which are applicable for patients is frequently palliative rather than curative. This has to suicide gene therapy (reviewed in Refs 6, 7). These can be categoprompted the development of several alternative, so called ‘biologi- rized as: (1) physical methods for disrupting the target cell memcal’ strategies (reviewed in Refs 1–5), one of which is somatic gene brane by mechanical or electrical means; (2) chemical methods that therapy. This discipline combines molecular biology, classical gen- employ a variety of non-liposome and liposome-based facilitators; etics and pharmacotherapeutics to manipulate the genomic compos- (3) recombinant viral vectors developed from early systems that are ition of a target cell with the aim of correcting an inherited or ac- based on manipulation of the papilloma simian virus SV40; and (4) quired defect. The potential range of disorders that is amenable to packaging cell lines that have been engineered to create a chimeric this form of therapy is vast, resulting in the development of different genome by incorporating genes that encode for viral envelope prostrategies. Therapeutic efficacy is dependent on judicious consider- teins. Transfection with a vector construct that contains the approation of several factors that are common to all of these strategies. priate long terminal repeats (LTRs), packaging sequence and suicide These include: (1) choice of target cell type(s); (2) a suitable tissue- gene (Fig. 1) results in the formation of virions that are then used to specific delivery system; (3) high transfection rate; (4) a suitable infect target cells. 0167-5699/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
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Table 1. Methods and vehicles for gene delivery Optimizing gene delivery A major problem in gene delivery is that the methods described above have characteristics, each of which can present as either an obstacle, or an aid, to gene transfer. This idiosyncrasy depends on the size of the DNA construct, whether transient or sustained expression of the gene is required, the anatomical site(s) of the lesion, target tissue type, whether the transfer is to be carried out in vitro, in vivo or ex vivo, and vector type (Table 2). Thus, hybridization of these techniques has been necessary for optimal delivery of the therapeutic gene in given pathologies. This is illustrated in the following examples.
Category
Methods and vehicles
Refs
Physical
Direct intramuscular injection of plasmid DNA Direct intracellular microinjection Electroporation Jet injection Particle bombardment (biolistics)
6, 7 6, 7 6, 7 6, 7 6, 7
Chemical
DEAE-dextran Calcium phosphate co-precipitation DNA-polylysine-cell receptor conjugates Polybrene-DMSO Liposome-mediated DNA transfer
6, 7 6, 7 6, 7 6, 7 6, 7
Recombinant viral vector systems
Papilloma simian virus SV40 Retroviral vectors: avian spleen necrosis retrovirus Harvey murine sarcoma virus Moloney leukaemia murine virus Adenoviruses Adeno-associated viruses Lentiviruses: human immunodeficiency virus simian immunodeficiency virus feline immunodeficiency virus Polyoma virus Vaccinia virus Sindbis and Semliki Forest virus Sendai virus
8
Receptor-mediated gene delivery One approach uses the physiological capacity of cells to internalize macromolecules through receptor-mediated endocytosis. The choice of receptor serves in targeting delivery of the DNA. Negatively charged DNA is bound to the polycationic amine polylysine (PL), resulting in condensation of the DNA into a compact toroidal structure. This conjugate, when bound to a receptor-specific Packaging cell lines ligand, forms a complex that can bind to the receptor and be internalized via endocytosis. This approach has been used extensively. The asialoglycoprotein (ASGP) cell surface receptor is unique to hepatocytes and has been used to demonstrate cell-specific delivery of ASGP–PL–DNA complexes both in vitro and in vivo25. The transferrin receptor has also been used to mediate the delivery of transferrin–PL–DNA complexes into haematopoietic cells, human leukaemic cells and respiratory epithelial cells. Although internalization is efficient, the level of gene expression is highly variable owing to lysosomal degradation of the conjugate vectors. Several viruses infect cells via receptor-mediated endocytosis but are able to escape this degradation. The adenovirus disrupts the endosomal membrane by acidification. Simultaneous delivery of transferrin– PL–DNA conjugates and adenovirus to HeLa cells results in a threefold increase in gene expression compared with delivery of the conjugates alone; this is due to reduced lysosomal degradation and increased transduction frequency (from ,1% to .90%) in the target cell population26. The next logical step in this approach was to develop a means of incorporating the adenovirus into the conjugate without impeding its binding to the cell surface receptor or its ability for endosomal disruption27. Use of the Escherichia coli lacZ gene, which encodes the reporter enzyme b-galactosidase (conferring blue staining upon introduction of the substrate X-gal), has demonstrated that delivery of as few as ten DNA molecules per cell can produce detectable gene
9 10 11 12, 13, 14 15 16 17 18 19 20 21 22
NIH-3T3 mouse fibroblasts 293-human embryonic kidney cell line
23 24
expression. This is an astonishing 50 000-fold less DNA required than for other DNA-mediated transfer techniques, indicating that in some way the adenovirus aids the delivery of DNA to the nucleus. A worrying aspect of using adenovirus in this way is that the receptor
5´ Flanking region ψ
Packaging genes 3´ Flanking region
Enhancer/ promoter sequence
Poly-A sequence Immunology Today
Fig. 1. Essential elements for replication in a viral vector. For gene delivery, the packaging genes (E sequences in the adenoviral family, or gag, pol and rev in retroviruses) are replaced by the therapeutic gene, leaving the packaging sequence (c) intact but rendering the virus replication-defective. The packaging genes are inserted into the genome of a cell, which can then supply, in trans, the protein products when transfected with the mutated virus.
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Table 2. Pseudotyping of retroviruses according to host species preferencea Host species
Retrovirus type
Mammalian cell types
Polytropic and amphotropic Ecotropic Xenotropic
Murine cells and rat cells only Most other species, excluding murine a
This preferential infectivity is dependent on the specific glycoprotein encoded by the env gene in the retrovirus.
is produced in a wide variety of cell types making specific target delivery problematic. This has been circumvented by the use of ligand-specific rather than adenovirus-specific receptors after the endosomolytic ability of the virus was shown to be independent of its mode of entry into the cell6. Another aspect that raises concern is that most of the human population carries adenoviral antibodies as a result of previous infections from the wild-type virus. Because the adenoviral vectors that are used have been rendered replication-incompetent and because transfected cells are killed by cytotoxic T-lymphocytes (CTLs), in vivo administration of these vectors has resulted in a high but transient (5–10 days post-infection) gene expression. The synthesis of a so-called ‘gutless’ vector, in which all of the genes have been deleted except those adenoviral sequences that encode the packaging sequence and the elements that define the beginning and end of the genome, has resulted in a much more sustained expression (up to 84 days post-infection)28.
Viral pseudotyping for targeted gene delivery Another approach in creating optimized vectors is the so-called pseudotype technique in which viral vector particles are manipulated
to incorporate viral glycoproteins of different origins. This technique has been employed principally for the construction of retroviral pseudotypes that exhibit extended species and tissue tropism29, and that might thus also be used as vehicles for the delivery of genes of interest (e.g. suicide genes) in a highly specific manner to selected human target cells and tissues. This was demonstrated by Spiegel et al.30 through retroviral pseudotype targeting of the liver-specific ASGP receptor (ASGP-R). Recombinant ecotropic retroviral Moloney murine leukaemia virus particles pseudotyped with Sendai virus F-glycoprotein [MoMLV(SeV-F)] have been found to transduce ASGP-R1 human hepatoma cells, but not those that are ASGP-R2. MoMLV(SeV-F) pseudotype vectors might thus emerge as prime candidates for specific and restricted transduction of ASGP-R1 cells in, for example, suicide gene therapy of human hepatomas.
Development of suicide gene therapy Originally, the suicide gene was conceptualized to act first, as a failsafe mechanism in retroviral vectors and second, as a phenotypic marker for scoring transfection rates. The promiscuous ability of retroviruses to infect a variety of cell types made them popular vectors for gene transfer. However, the random nature of their integration into host chromosomes has led to fears that the promoter element might cause downstream activation of an oncogene or a gene that encodes for some growth factor, leading to uncontrolled cellular proliferation. A gene that can be selectively switched on to encode for an enzyme that has no direct effect on cellular function but is capable of conferring toxicity to an otherwise benign compound (prodrug) has been incorporated into the vector as a supplement to the ‘therapeutic’ gene. Introduction of the prodrug prompts those cells that carry this supplementary gene to synthesize the enzyme and thus bring about their own death (Fig. 2). Hence, the era of the suicide gene was born.
HSV-tk
Fig. 2. The sequence of events, from incorporation of the suicide gene into the host cell genome via a suitable delivery vehicle, activation by the appropriate prodrug and eventual cell death. This gene encodes for an enzyme that converts the inactive prodrug into its toxic metabolites. These act by inhibition of DNA or RNA synthesis, or both, and hence cell growth and division.
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A short but crucially important leap of imagination has shifted the role of the suicide gene from being supplementary to that of being primarily therapeutic. It has been reasoned that if activating the suicide gene prevents vector-induced iatrogenic tumours from transfected cells, then the same gene, inserted into the genome of cells that have acquired tumorigenicity through some other mishap, should also compel these cells to commit suicide upon introduction of the prodrug. Initial demonstrations have revealed that transfer of the gene for herpes simplex virus thymidine kinase (HSV-tk) into mammalian normal and malignant cells followed by treatment with the anti-herpes drugs acyclovir (ACV) or ganciclovir (GCV) leads to selective death of transduced cells, both in vitro and in vivo32.
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Box 1. Future requirements and developments in suicide gene therapy GCV is the more effective of the two. The enzymic phosphorylation of GCV by HSV-tk to its diphosphate and triphosphate metabolites confers cytotoxicity by inhibiting the action of DNA polymerase.
Cytosine deaminase Another widely used suicide gene encodes the bacterial and fungal enzyme cytosine deaminase (CDA), which catalyzes the hydrolytic deamination of cytosine to uracil. This metabolic pathway has been exploited in the use of 5-fluorocytosine (5-FC) as a fungicidal and bactericidal drug. Microorganisms that express the gene for CDA convert 5-FC to 5-fluorouracil (5-FU), which is then phosphorylated to form 5-FU monophosphate and 5-FU triphosphate. These inhibit thymidilate synthetase and mRNA transcription, respectively. This interference of DNA and RNA synthesis results in cell death. Because mammalian cells do not contain significant amounts of CDA, 5-FC is relatively non-toxic, but insertion of this gene promotes 5-FC to the position of a prodrug. This was first demonstrated in three murine cell lines by Mullen et al.33 who, using a replicationincompetent retroviral vector for transduction, inserted the E. coli gene for CDA into NIH-3T3 fibroblasts, murine sarcoma cells (line 207-10) and murine colon adenocarcinoma cells (line 38-2). The first in vitro demonstration in human cells followed shortly. Austin and Huber34 have inserted the gene encoding CDA into WiDr cells (a human colorectal cancer cell line) and have shown a 560-fold increase in the toxicity of 5-FC.
Other suicide genes Other suicide genes have been used to potentiate chemotherapeutic agents in the treatment of cancers. Deoxycytidine kinase (dCK) phosphorylates several nucleoside analogues in use as anti-cancer drugs. Insertion of the gene for dCK into MCF-7 cells (a glioma cell line) results in a 2.5-fold increase in the toxicity of the anti-metabolites cytarabine, cytosine arabinoside, cladribine and 2-chlorodeoxyadenosine35. Following transfection with the gene for cytochrome P-450, an increased sensitivity of MCF-7 cells and another glioma cell line (9L) to the oxazaphosphorines cyclophosphamide and ifosfamide, both in vitro and after implantation in nude mice, has also been demonstrated36. Further roles for suicide genes have been proposed; some are putative, some are under active investigation and others are in clinical use (Box 1). These include: (1) their use in vaccinations using live unattenuated virus, bacteria or protozoa; (2) the controlled elimination of transfected target tissues in developmental studies; (3) a means for controlling the duration of treatment of a disorder by a co-packaged therapeutic gene; and (4) the purging of selected cell populations in therapies that involve bone marrow transplantation (BMT). These are described later.
Pre-clinical studies in mouse and rat models The formulation of a hypothesis from the initial flash of inspiration requires validation in the laboratory before any clinical application.
• Development of relevant disease models in higher vertebrates with physiological traits that are closer to humans • Development of new delivery systems, as well as the optimization of existing ones • Development of tissue and cell-type-specific promoter/ enhancer elements to achieve increased gene expression • Development of more versatile marker genes to monitor vector targeting and therapeutic progress • Development of a new speciality that would investigate the pharmacokinetics of gene delivery and expression • Further investigations are required into ways of enhancing the bystander effect. Examples of current work include investigations into connexin 43 gap junctions and the pharmacological enhancement of membrane permeability with hydroxyurea • Modulation of the host immune response to enhance and complement suicide gene therapy. Knowledge in this area is sparse, and investigations are urgently required for greater treatment efficacy • Developments are required in coupling chimeric suicide gene constructs to multidrug resistance genes, allowing a combined approach using the suicide gene together with reduced, and thus more palatable, doses of conventionally used cytotoxic drugs • Further exploration is required into the phenomenon of certain suicide genes behaving as radiosensitizing agents. This could lead to a combined approach of suicide gene therapy with radiation therapy, but with lower and better targeted doses of radiation • Further exploration is required into the effects of including heat shock protein promotional sequences into suicide gene constructs. The suicide gene can then be induced by localized application of heat, providing a unique application for hyperthermia in cancer therapy The use of animal models that phenotypically mirror manifestations of the human disease in question is essential. Scientific literature is replete with mouse and rat models of human disorders and, if inherent physiological differences are borne in mind, provide a convenient and relatively cheap vehicle for pre-clinical studies (Table 3). Information that is yielded by these animals in gene targeting has been invaluable. To describe all these studies adequately is beyond the scope of this article, but we present the following illustrative examples. One of the earliest demonstrations of tumoricidal effects of CDA was in rats bearing subcutaneous glioma cell tumours37. The purified enzyme was placed in dialysis tubing and implanted adjacent to the tumours. A reduction in tumour size was observed following treatment with 5-FC for 30 days. Culver et al.14 pioneered the use of the gene for HSV-tk in cancer therapy. They injected vector-producing cells (after transduction with the HSV-tk retroviral vector) directly into sarcoglioma tumours in rat brains. Treatment with GCV resulted in complete regression in
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11 out of 14 rats, even when only 10% of the Table 3. Examples of mutagen-induced diseases in the murine model tumour cells were infected. This was exDeficiency phenotype Target tissue plained by the ‘bystander effect’ whereby Human disease in the murine model or cell type toxic nucleotides that are generated within infected cells are transmitted via gap juncPhenylketonuria Phenylalanine hydroxylase Liver tions to adjacent non-infected cells. The byDuchenne muscular dystrophy Dystrophin Satellite cells in stander effect also occurs with the CDA suiskeletal muscle cide gene, although the mechanism differs in Adenomatous intestinal polyposis Adenomatous polyposis Colon epithelial that 5-FU is freely diffusable across cell coli protein cells membranes. The outcome in both is that Gaucher’s disease Glucocerebrosidase Haematopoietic non-infected, but bystander-toxified cells are stem cells, killed. macrophages Cystic fibrosis Cystic fibrosis Nasal and bronchial In vivo transduction levels can be low, transmembrane epithelium particularly in solid tumours (e.g. in larger conductance protein hepatoma nodules), limiting therapeutic a- and b-thalassaemias Haemoglobin a- and Erythroid progenitor efficacy, even in the presence of a strong byb-chains, respectively cells stander effect. Potentiation of the bystander Glucose-6-phosphate Glucose-6-phosphate Liver effect is highly desirable. Recent developdehydrogenase deficiency dehydrogenase ments have capitalized on the remarkable inAtherosclerosis and Apolipoprotein E Vascular endothelial tercellular transport properties of the struchypercholesterolaemia cells and liver, tural protein VP22 of HSV-1 (Ref. 38). After respectively in vitro expression in a subpopulation of Spontaneous tumour formation p53 protein Varied: dependent cells, VP22 has been found to spread to 100% on tumour origin of cells in a monolayer culture. Interestingly, T- and B-cell immunodeficiencies V(D)J recombination Lymphoid these intercellular trafficking properties also activating protein progenitor cells apply to VP22 fusion proteins. Because the Adenosine deaminase-induced Adenosine deaminase T cells, bone delivery of therapeutic genes or gene prodsevere combined marrow stem cells ucts into sufficient numbers of tumour cells immunodeficiency (SCID/ADA) remains a major difficulty, the use of VP22 fusion proteins could significantly increase the percentage of targeted cells. Furthermore, a recent study by possibly owing to the bacterial CDA protein functioning as a superWybranietz et al.31 has demonstrated that spread of VP22 fusion pro- antigen, resulting in polyclonal activation of T-lymphocytes. Subteins is a general biological phenomenon that is not restricted to dis- sequent studies using tumour cell lines that produced both CDA and tinct tissues or species. In principle, this approach could easily be IL-6 showed a failure to establish tumours upon subcutaneous injecadapted for potentiation of the bystander effect in solid tumours by tion in immunocompetent mice. It was thought that T cells and natural generation of VP22–HSV-tk or VP22–CDA fusion genes. killer (NK) cells were responsible for a majority of the activity and The use of interleukin 2 (IL-2) as an adjunctive therapy to HSV-tk that IL-6 might have been enhancing the T-cell response or antigen expression has been explored by Chen et al.39 Tumours were pro- presentation by B cells. duced in BALB/c mice by intraperitoneal injection of a colon cancer Santodonato et al.42 have combined the use of vaccination and cell line (MCA26). HSV-tk and IL-2 were delivered using two adeno- gene therapy with HSV-tk. Metastatic Friend erythroleukaemia cell viral vectors. GCV treatment resulted in large-scale tumour necrosis, (FLC) tumours are suppressed following vaccination with interferon with three out of the ten animals tumour-free. Significantly, an ap- a (IFN-a)-producing FLCs. Using IFN-a-producing FLC lines that preciable CTL response against the MCA26 cells was seen only in have been transfected with the gene for HSV-tk, a 70–100% cure rate those mice receiving both HSV-tk and IL-2. A further study using can be demonstrated in mice, with no secondary tumour formation. combined HSV-tk–IL-2 therapy in a murine oral cancer model, These mice are resistant to subsequent challenge with the wild-type showed a median survival time of three weeks compared with two parental tumour cells. High-dose chemotherapy with autologous BMT is an important weeks in mice that received HSV-tk alone, and one week in those treatment for patients with breast cancer. Purging techniques are rewith lacZ as a control gene40. Mullen et al.41, using the CDA–5-FC suicide system, transfected quired to limit potential contamination of the autologous graft by weakly immunogenic C57BL/6-derived colon cancer and fibrosar- residual tumour cells. Selective targeting of these tumour cells with coma cell lines and subcutaneously injected these cells into syn- a suicide-gene-containing vector offers a promising approach. Feasigeneic mice. Treatment with 5-FC resulted in substantial tumour bility tests in various mouse transplantation models are currently regression. Significantly, a reduction in secondary tumour formation under way. In one, Nanakorn et al.43, using adenoviral-vector–CDA upon challenge with syngeneic wild-type tumour cells was noted, gene targeting of breast cancer cells (BCCs) in mixtures of BCCs and
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haematopoietic cells, have shown sustained engraftment after 14 days of 5-FC treatment post-transplant. They now propose that peripheral blood stem cells from breast cancer patients undergoing autografts should be exposed in vitro to the adenoviral vector, followed by transplantation and a 14-day 5-FC treatment for in vivo purging of BCCs.
Human studies and clinical trials Ethical approval was given for the first gene therapy clinical trial as recently as 1990 and over 300 more such trials have been initiated. Although a majority of these trials are in Phase I/II stage, it is indicative of the rate at which this field is progressing and the urgency for this therapy. Over half of these trials involve therapeutic approaches to malignant disorders with approximately 40 for the correction of monogenic diseases such as severe combined immunodeficiency–adenosine deaminase deficiency (SCID–ADA), cystic fibrosis and a1-antitrypsin deficiency. The full scope is even wider, covering disorders that range from atherosclerosis to arthritis and Duchenne muscular dystrophy. Suicide genes have been used both as a safety mechanism secondary to the therapeutic gene and as the primary therapeutic gene indicating their importance and versatility in gene therapy. Lack of space does not allow a detailed account of these trials, but a few examples are offered. The first clinical trial to use a suicide gene as the primary therapeutic agent was approved in 1991 (Ref. 44) for the treatment of ovarian carcinoma. Ovarian cancer cells were transfected ex vivo with the gene for HSV-tk and then instilled intraperitoneally in nine patients in stage III of the disease. One patient achieved complete remission, whereas others showed partial tumour regression. It is arguable that use of more efficient methods of delivery that have been developed since would lead to better results. BMT is a potentially curative procedure in selected patients with haematological malignancies. Graft–versus–host disease (GVHD) remains a major problem following non-T-cell-depleted allogeneic BMT. Although donor T cells play a central role in both the graft–versus–leukaemia effect and immune reconstitution, the individual risk for GVHD cannot be predicted; ex-vivo transduction of these cells with a suicide gene before BMT offers a promising means for eliminating them at will. This is an approach that is being followed, for example with the truncated form of the low-affinity nerve growth factor gene (DLNGRF)–CDA–5-FC suicide system in a paediatric cohort. Bordignon et al.45, using a bicistronic retroviral vector, have transferred the HSV-tk gene and DLNGRF gene into allogeneic donor PBLs. The expression product of the DLNGRF gene was used as a marker for selection and enumeration of transduced cells. A total of 12 patients who suffered from severe complications following alloBMT or immunodeficiency were enrolled. A follow-up report has been published on eight of these patients46 revealing that three developed GVHD. Treatment with GCV resulted in elimination of transduced lymphocytes and GVHD, confirming the therapeutic potential of suicide gene therapy in the context of allo-BMT and GVHD. Several trials are under way for the treatment of malignant gliomas using the suicide gene approach and early results are
indicative of anti-tumour responses in several patients47. The brain deserves a special mention as an example of a tissue with relatively low immunological background, and low level of cell division. These features must be considered when devising strategies for gene therapy in this organ. An inherent advantage is that long-term gene expression is not required to effect cell death. Furthermore, the use of retroviral vectors provides selective delivery of the suicide gene to actively dividing tumour cells. Delivery can be enhanced further by cytokine stimulation of tumour cell growth. Results from these trials are awaited with anticipation.
Concluding remarks The need for gene therapy is paramount and the role of suicide genes as primary therapeutic agents and as safety switches in the delivery of other genes has been established. Although encouraging results are emerging from pre-clinical and clinical studies, the need for more efficient and specific methods of delivery are constantly being highlighted. Promising new avenues of research point to the eventual dovetailing of suicide gene therapy with more traditional therapies, resulting in novel techniques for patient care (Box 1). The dismal prognosis in many disorders acts as a spur in finding a solution to these problems. Opportunities for tackling existing and emerging diseases through gene manipulation abound. We are optimistic that, in the not too distant future, suicide gene therapy will complement, and even supercede some of the more conventional modalities. Indeed, the art of suicide gene therapy will itself become a convention.
We thank J. Wessels for help in preparing the diagrams and M. Eyrich for comments and suggestions. S.L. is the recipient of a Graduate Student Fellowship from the fortüne Foundation of the University Clinic of Tübingen. We acknowledge support from the fortüne Program (grants #375/1996 and #438/1997), the Deutsche Forschungsgemeinschaft (grants SFB510-C4 and La649/11-1), the Federal Ministry of Education, Science, Research and Technology (grant Fö. 01KS9602), the Interdisciplinary Research Centre (IKFZ) Tübingen and the Deutsche José Carreras Leukaemie-Stiftung (grant #DJCLS-R21).
Shangara Lal ([email protected]), Dietrich Niethammer and Paul Schlegel ([email protected]) are at the Dept of Paediatric Haematology and Oncology, Children’s Hospital, University of Tübingen, D-72076 Tübingen, Germany; Ulrich Lauer is at the Dept of Internal Medicine, University of Tübingen, D-72076 Tübingen, Germany; James Beck is at the University Children’s Hospital, Ernst-Moritz-Arndt University of Greifswald, D-17487 Greifswald, Germany.
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28 Chen, H.H. et al. (1997) Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc. Natl. Acad. Sci. U. S. A. 94, 1645–1650 29 Burns, J.C. et al. (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to a very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. U. S. A. 90, 8033–8037 30 Spiegel, M. et al. (1998) Pseudotype formation of Moloney murine leukemia virus with Sendai virus glycoprotein F, J. Virol. 72, 5296–5302 31 Wybranietz, W.A. et al. (1999) Quantification of VP22–GFP spread by direct fluorescence in 15 commonly used cell lines. J. Gene Med. 1, 265–274 32 Heyman, R.A. et al. (1989) Thymidine kinase obliteration: creation of transgenic mice with controlled immune deficiency. Proc. Natl. Acad. Sci. U. S. A. 86, 2698–2702 33 Mullen, C.A. et al. (1992) Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5fluorocytosine: a negative selection system. Proc. Natl. Acad. Sci. U. S. A. 89, 33–37 34 Austin, E.A. and Huber, B.E. (1992) A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol. Pharmacol. 43, 380–387 35 Hapke, D.M. et al. (1996) Retroviral transfer of deoxycytidine kinase into tumor cell lines enhances nucleoside toxicity. Cancer Res. 56, 2343–2347 36 Chen, L. et al. (1996) Sensitization of human breast cancer cells to cyclophosphamide and ifosfamide by transfer of a liver cytochrome P450 gene. Cancer Res. 56, 1331–1340 37 Nishiyama, T. et al. (1985) Antineoplastic effects in rats of 5-fluorocytosine in combination with cytosine deaminase capsules. Cancer Res. 45, 1753–1761 38 Phelan, A. et al. (1998) Intercellular delivery of functional p53 by the herpesvirus protein VP22. Nat. Biotechnol. 16, 440–443 39 Chen, S.H. et al. (1995) Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc. Natl. Acad. Sci. U. S. A. 92, 2577–2581 40 O’Malley, B.W.J. et al. (1996) Combination gene therapy for oral cancer in a murine model. Cancer Res. 56, 1737–1741 41 Mullen, C.A. et al. (1994) Tumors expressing the cytosine deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild type tumor. Cancer Res. 54, 1503–1506 42 Santodonato, L. et al. (1996) Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-a1 and herpes simplex thymidine kinase followed by ganciclovir. Hum. Gene Ther. 7, 1–10 43 Nanakorn, T. et al. (1997) Cytosine deaminase adenoviral vector system reduces levels of contaminating breast cancer cells admixed with a thousand fold excess of hematopoietic cells by up to 500,000 fold following exposure to 5-fluorocytosine, which is converted to 5-fluorouracil by this system selectively in the breast cancer cells. Blood 90 (Suppl. 1), 555a 44 Freeman, S.M. et al. (1995) Treatment of ovarian cancer using a genemodified vaccine. Hum. Gene Ther. 6, 927–939 45 Bordignon, C. et al. (1995) Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor antitumor immunity after allogeneic bone marrow transplantation. Hum. Gene Ther. 6, 813–819 46 Bonini, C. et al. (1997) HSV-tk gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276, 1719–1724 47 Freeman, S.M. et al. (1996) In situ use of suicide genes for cancer therapy. Semin. Oncol. 23, 31–45
53 Burlingham, W.J. (1996) Chimerism after organ transplantation: is there any clinical significance? Clin. Transplant. 10, 110–117 54 Kimikawa, M. et al. (1997) Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys. Transplantation 64, 709–716 55 Greenstein, J.L. and Sachs, D.H. (1997) The use of tolerance for transplantation across xenogeneic barriers. Nat. Biotechnol. 15, 235–238 56 Sablinski, T. et al. (1997) Pig to monkey bone marrow and kidney xenotransplantation. Surgery 121, 381–391 57 Naji, A. (1996) Induction of tolerance by intrathymic inoculation of alloantigen. Curr. Opin. Immunol. 8, 704–709
58 Allen, M.D. et al. (1997) Prolonged allogeneic and xenogeneic microchimerism in unmatched primates without immunosuppression by intrathymic implantation of CD341 donor marrow cells. Cell. Immunol. 181, 127–138 59 Groth, C.G. et al. (1994) Transplantation of porcine fetal pancreas to diabetic patients. Lancet 344, 1402–1404 60 Paradis, K. et al. (1999) Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 285, 1236–1241 61 Patience, C. et al. (1997) Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3, 282–286
Suicide genes: past, present and future perspectives Shangara Lal, Ulrich M. Lauer, Dietrich Niethammer, James F. Beck and Paul G. Schlegel Disappointing morbidity and mortality rates in many disorders
‘I
f one considers the purpose of a promoter/enhancer sequence for sustained have necessitated the development of drug to be to restore normal funcand optimal gene expression; and (5) a means several ‘biological’ therapies. One tion of some particular process in for controlling the level of gene expression. approach involves the use of suicide the body, then DNA would be Suicide gene therapy is one such strategy considered to be the ultimate drug…’ The that uses the principle of inserting a sogenes. Here, Shangara Lal and foundations of molecular medicine rest called ‘suicide gene’ into the target genome. colleagues discuss the development squarely on Watson and Crick’s discovery of This gene encodes for an enzyme that conof this branch of gene therapy in cell the structure and function of DNA. This verts an inactive prodrug into its cytotoxic and the subsequent discovery of mRNA has metabolite(s) so that, upon introduction of lines and animal models, and led to the formulation of the ‘central dogma’ the prodrug, the target cell effectively is examine current studies, therapeutic that dictates the flow of genetic information compelled to commit suicide. applications and clinical trials. from DNA to RNA to protein. Aberrations along this pathway can manifest as clinical Evolving methods of gene delivery disorders. Morbidity and mortality rates for a variety of malignant cancers, autoimmune and metabolic disorders Several techniques have been developed for transferring genes have only marginally improved over the past 15 or so years and care across the cell membrane (Table 1), many of which are applicable for patients is frequently palliative rather than curative. This has to suicide gene therapy (reviewed in Refs 6, 7). These can be categoprompted the development of several alternative, so called ‘biologi- rized as: (1) physical methods for disrupting the target cell memcal’ strategies (reviewed in Refs 1–5), one of which is somatic gene brane by mechanical or electrical means; (2) chemical methods that therapy. This discipline combines molecular biology, classical gen- employ a variety of non-liposome and liposome-based facilitators; etics and pharmacotherapeutics to manipulate the genomic compos- (3) recombinant viral vectors developed from early systems that are ition of a target cell with the aim of correcting an inherited or ac- based on manipulation of the papilloma simian virus SV40; and (4) quired defect. The potential range of disorders that is amenable to packaging cell lines that have been engineered to create a chimeric this form of therapy is vast, resulting in the development of different genome by incorporating genes that encode for viral envelope prostrategies. Therapeutic efficacy is dependent on judicious consider- teins. Transfection with a vector construct that contains the approation of several factors that are common to all of these strategies. priate long terminal repeats (LTRs), packaging sequence and suicide These include: (1) choice of target cell type(s); (2) a suitable tissue- gene (Fig. 1) results in the formation of virions that are then used to specific delivery system; (3) high transfection rate; (4) a suitable infect target cells. 0167-5699/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
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Table 1. Methods and vehicles for gene delivery Optimizing gene delivery A major problem in gene delivery is that the methods described above have characteristics, each of which can present as either an obstacle, or an aid, to gene transfer. This idiosyncrasy depends on the size of the DNA construct, whether transient or sustained expression of the gene is required, the anatomical site(s) of the lesion, target tissue type, whether the transfer is to be carried out in vitro, in vivo or ex vivo, and vector type (Table 2). Thus, hybridization of these techniques has been necessary for optimal delivery of the therapeutic gene in given pathologies. This is illustrated in the following examples.
Category
Methods and vehicles
Refs
Physical
Direct intramuscular injection of plasmid DNA Direct intracellular microinjection Electroporation Jet injection Particle bombardment (biolistics)
6, 7 6, 7 6, 7 6, 7 6, 7
Chemical
DEAE-dextran Calcium phosphate co-precipitation DNA-polylysine-cell receptor conjugates Polybrene-DMSO Liposome-mediated DNA transfer
6, 7 6, 7 6, 7 6, 7 6, 7
Recombinant viral vector systems
Papilloma simian virus SV40 Retroviral vectors: avian spleen necrosis retrovirus Harvey murine sarcoma virus Moloney leukaemia murine virus Adenoviruses Adeno-associated viruses Lentiviruses: human immunodeficiency virus simian immunodeficiency virus feline immunodeficiency virus Polyoma virus Vaccinia virus Sindbis and Semliki Forest virus Sendai virus
8
Receptor-mediated gene delivery One approach uses the physiological capacity of cells to internalize macromolecules through receptor-mediated endocytosis. The choice of receptor serves in targeting delivery of the DNA. Negatively charged DNA is bound to the polycationic amine polylysine (PL), resulting in condensation of the DNA into a compact toroidal structure. This conjugate, when bound to a receptor-specific Packaging cell lines ligand, forms a complex that can bind to the receptor and be internalized via endocytosis. This approach has been used extensively. The asialoglycoprotein (ASGP) cell surface receptor is unique to hepatocytes and has been used to demonstrate cell-specific delivery of ASGP–PL–DNA complexes both in vitro and in vivo25. The transferrin receptor has also been used to mediate the delivery of transferrin–PL–DNA complexes into haematopoietic cells, human leukaemic cells and respiratory epithelial cells. Although internalization is efficient, the level of gene expression is highly variable owing to lysosomal degradation of the conjugate vectors. Several viruses infect cells via receptor-mediated endocytosis but are able to escape this degradation. The adenovirus disrupts the endosomal membrane by acidification. Simultaneous delivery of transferrin– PL–DNA conjugates and adenovirus to HeLa cells results in a threefold increase in gene expression compared with delivery of the conjugates alone; this is due to reduced lysosomal degradation and increased transduction frequency (from ,1% to .90%) in the target cell population26. The next logical step in this approach was to develop a means of incorporating the adenovirus into the conjugate without impeding its binding to the cell surface receptor or its ability for endosomal disruption27. Use of the Escherichia coli lacZ gene, which encodes the reporter enzyme b-galactosidase (conferring blue staining upon introduction of the substrate X-gal), has demonstrated that delivery of as few as ten DNA molecules per cell can produce detectable gene
9 10 11 12, 13, 14 15 16 17 18 19 20 21 22
NIH-3T3 mouse fibroblasts 293-human embryonic kidney cell line
23 24
expression. This is an astonishing 50 000-fold less DNA required than for other DNA-mediated transfer techniques, indicating that in some way the adenovirus aids the delivery of DNA to the nucleus. A worrying aspect of using adenovirus in this way is that the receptor
5´ Flanking region ψ
Packaging genes 3´ Flanking region
Enhancer/ promoter sequence
Poly-A sequence Immunology Today
Fig. 1. Essential elements for replication in a viral vector. For gene delivery, the packaging genes (E sequences in the adenoviral family, or gag, pol and rev in retroviruses) are replaced by the therapeutic gene, leaving the packaging sequence (c) intact but rendering the virus replication-defective. The packaging genes are inserted into the genome of a cell, which can then supply, in trans, the protein products when transfected with the mutated virus.
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Table 2. Pseudotyping of retroviruses according to host species preferencea Host species
Retrovirus type
Mammalian cell types
Polytropic and amphotropic Ecotropic Xenotropic
Murine cells and rat cells only Most other species, excluding murine a
This preferential infectivity is dependent on the specific glycoprotein encoded by the env gene in the retrovirus.
is produced in a wide variety of cell types making specific target delivery problematic. This has been circumvented by the use of ligand-specific rather than adenovirus-specific receptors after the endosomolytic ability of the virus was shown to be independent of its mode of entry into the cell6. Another aspect that raises concern is that most of the human population carries adenoviral antibodies as a result of previous infections from the wild-type virus. Because the adenoviral vectors that are used have been rendered replication-incompetent and because transfected cells are killed by cytotoxic T-lymphocytes (CTLs), in vivo administration of these vectors has resulted in a high but transient (5–10 days post-infection) gene expression. The synthesis of a so-called ‘gutless’ vector, in which all of the genes have been deleted except those adenoviral sequences that encode the packaging sequence and the elements that define the beginning and end of the genome, has resulted in a much more sustained expression (up to 84 days post-infection)28.
Viral pseudotyping for targeted gene delivery Another approach in creating optimized vectors is the so-called pseudotype technique in which viral vector particles are manipulated
to incorporate viral glycoproteins of different origins. This technique has been employed principally for the construction of retroviral pseudotypes that exhibit extended species and tissue tropism29, and that might thus also be used as vehicles for the delivery of genes of interest (e.g. suicide genes) in a highly specific manner to selected human target cells and tissues. This was demonstrated by Spiegel et al.30 through retroviral pseudotype targeting of the liver-specific ASGP receptor (ASGP-R). Recombinant ecotropic retroviral Moloney murine leukaemia virus particles pseudotyped with Sendai virus F-glycoprotein [MoMLV(SeV-F)] have been found to transduce ASGP-R1 human hepatoma cells, but not those that are ASGP-R2. MoMLV(SeV-F) pseudotype vectors might thus emerge as prime candidates for specific and restricted transduction of ASGP-R1 cells in, for example, suicide gene therapy of human hepatomas.
Development of suicide gene therapy Originally, the suicide gene was conceptualized to act first, as a failsafe mechanism in retroviral vectors and second, as a phenotypic marker for scoring transfection rates. The promiscuous ability of retroviruses to infect a variety of cell types made them popular vectors for gene transfer. However, the random nature of their integration into host chromosomes has led to fears that the promoter element might cause downstream activation of an oncogene or a gene that encodes for some growth factor, leading to uncontrolled cellular proliferation. A gene that can be selectively switched on to encode for an enzyme that has no direct effect on cellular function but is capable of conferring toxicity to an otherwise benign compound (prodrug) has been incorporated into the vector as a supplement to the ‘therapeutic’ gene. Introduction of the prodrug prompts those cells that carry this supplementary gene to synthesize the enzyme and thus bring about their own death (Fig. 2). Hence, the era of the suicide gene was born.
HSV-tk
Fig. 2. The sequence of events, from incorporation of the suicide gene into the host cell genome via a suitable delivery vehicle, activation by the appropriate prodrug and eventual cell death. This gene encodes for an enzyme that converts the inactive prodrug into its toxic metabolites. These act by inhibition of DNA or RNA synthesis, or both, and hence cell growth and division.
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A short but crucially important leap of imagination has shifted the role of the suicide gene from being supplementary to that of being primarily therapeutic. It has been reasoned that if activating the suicide gene prevents vector-induced iatrogenic tumours from transfected cells, then the same gene, inserted into the genome of cells that have acquired tumorigenicity through some other mishap, should also compel these cells to commit suicide upon introduction of the prodrug. Initial demonstrations have revealed that transfer of the gene for herpes simplex virus thymidine kinase (HSV-tk) into mammalian normal and malignant cells followed by treatment with the anti-herpes drugs acyclovir (ACV) or ganciclovir (GCV) leads to selective death of transduced cells, both in vitro and in vivo32.
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Box 1. Future requirements and developments in suicide gene therapy GCV is the more effective of the two. The enzymic phosphorylation of GCV by HSV-tk to its diphosphate and triphosphate metabolites confers cytotoxicity by inhibiting the action of DNA polymerase.
Cytosine deaminase Another widely used suicide gene encodes the bacterial and fungal enzyme cytosine deaminase (CDA), which catalyzes the hydrolytic deamination of cytosine to uracil. This metabolic pathway has been exploited in the use of 5-fluorocytosine (5-FC) as a fungicidal and bactericidal drug. Microorganisms that express the gene for CDA convert 5-FC to 5-fluorouracil (5-FU), which is then phosphorylated to form 5-FU monophosphate and 5-FU triphosphate. These inhibit thymidilate synthetase and mRNA transcription, respectively. This interference of DNA and RNA synthesis results in cell death. Because mammalian cells do not contain significant amounts of CDA, 5-FC is relatively non-toxic, but insertion of this gene promotes 5-FC to the position of a prodrug. This was first demonstrated in three murine cell lines by Mullen et al.33 who, using a replicationincompetent retroviral vector for transduction, inserted the E. coli gene for CDA into NIH-3T3 fibroblasts, murine sarcoma cells (line 207-10) and murine colon adenocarcinoma cells (line 38-2). The first in vitro demonstration in human cells followed shortly. Austin and Huber34 have inserted the gene encoding CDA into WiDr cells (a human colorectal cancer cell line) and have shown a 560-fold increase in the toxicity of 5-FC.
Other suicide genes Other suicide genes have been used to potentiate chemotherapeutic agents in the treatment of cancers. Deoxycytidine kinase (dCK) phosphorylates several nucleoside analogues in use as anti-cancer drugs. Insertion of the gene for dCK into MCF-7 cells (a glioma cell line) results in a 2.5-fold increase in the toxicity of the anti-metabolites cytarabine, cytosine arabinoside, cladribine and 2-chlorodeoxyadenosine35. Following transfection with the gene for cytochrome P-450, an increased sensitivity of MCF-7 cells and another glioma cell line (9L) to the oxazaphosphorines cyclophosphamide and ifosfamide, both in vitro and after implantation in nude mice, has also been demonstrated36. Further roles for suicide genes have been proposed; some are putative, some are under active investigation and others are in clinical use (Box 1). These include: (1) their use in vaccinations using live unattenuated virus, bacteria or protozoa; (2) the controlled elimination of transfected target tissues in developmental studies; (3) a means for controlling the duration of treatment of a disorder by a co-packaged therapeutic gene; and (4) the purging of selected cell populations in therapies that involve bone marrow transplantation (BMT). These are described later.
Pre-clinical studies in mouse and rat models The formulation of a hypothesis from the initial flash of inspiration requires validation in the laboratory before any clinical application.
• Development of relevant disease models in higher vertebrates with physiological traits that are closer to humans • Development of new delivery systems, as well as the optimization of existing ones • Development of tissue and cell-type-specific promoter/ enhancer elements to achieve increased gene expression • Development of more versatile marker genes to monitor vector targeting and therapeutic progress • Development of a new speciality that would investigate the pharmacokinetics of gene delivery and expression • Further investigations are required into ways of enhancing the bystander effect. Examples of current work include investigations into connexin 43 gap junctions and the pharmacological enhancement of membrane permeability with hydroxyurea • Modulation of the host immune response to enhance and complement suicide gene therapy. Knowledge in this area is sparse, and investigations are urgently required for greater treatment efficacy • Developments are required in coupling chimeric suicide gene constructs to multidrug resistance genes, allowing a combined approach using the suicide gene together with reduced, and thus more palatable, doses of conventionally used cytotoxic drugs • Further exploration is required into the phenomenon of certain suicide genes behaving as radiosensitizing agents. This could lead to a combined approach of suicide gene therapy with radiation therapy, but with lower and better targeted doses of radiation • Further exploration is required into the effects of including heat shock protein promotional sequences into suicide gene constructs. The suicide gene can then be induced by localized application of heat, providing a unique application for hyperthermia in cancer therapy The use of animal models that phenotypically mirror manifestations of the human disease in question is essential. Scientific literature is replete with mouse and rat models of human disorders and, if inherent physiological differences are borne in mind, provide a convenient and relatively cheap vehicle for pre-clinical studies (Table 3). Information that is yielded by these animals in gene targeting has been invaluable. To describe all these studies adequately is beyond the scope of this article, but we present the following illustrative examples. One of the earliest demonstrations of tumoricidal effects of CDA was in rats bearing subcutaneous glioma cell tumours37. The purified enzyme was placed in dialysis tubing and implanted adjacent to the tumours. A reduction in tumour size was observed following treatment with 5-FC for 30 days. Culver et al.14 pioneered the use of the gene for HSV-tk in cancer therapy. They injected vector-producing cells (after transduction with the HSV-tk retroviral vector) directly into sarcoglioma tumours in rat brains. Treatment with GCV resulted in complete regression in
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11 out of 14 rats, even when only 10% of the Table 3. Examples of mutagen-induced diseases in the murine model tumour cells were infected. This was exDeficiency phenotype Target tissue plained by the ‘bystander effect’ whereby Human disease in the murine model or cell type toxic nucleotides that are generated within infected cells are transmitted via gap juncPhenylketonuria Phenylalanine hydroxylase Liver tions to adjacent non-infected cells. The byDuchenne muscular dystrophy Dystrophin Satellite cells in stander effect also occurs with the CDA suiskeletal muscle cide gene, although the mechanism differs in Adenomatous intestinal polyposis Adenomatous polyposis Colon epithelial that 5-FU is freely diffusable across cell coli protein cells membranes. The outcome in both is that Gaucher’s disease Glucocerebrosidase Haematopoietic non-infected, but bystander-toxified cells are stem cells, killed. macrophages Cystic fibrosis Cystic fibrosis Nasal and bronchial In vivo transduction levels can be low, transmembrane epithelium particularly in solid tumours (e.g. in larger conductance protein hepatoma nodules), limiting therapeutic a- and b-thalassaemias Haemoglobin a- and Erythroid progenitor efficacy, even in the presence of a strong byb-chains, respectively cells stander effect. Potentiation of the bystander Glucose-6-phosphate Glucose-6-phosphate Liver effect is highly desirable. Recent developdehydrogenase deficiency dehydrogenase ments have capitalized on the remarkable inAtherosclerosis and Apolipoprotein E Vascular endothelial tercellular transport properties of the struchypercholesterolaemia cells and liver, tural protein VP22 of HSV-1 (Ref. 38). After respectively in vitro expression in a subpopulation of Spontaneous tumour formation p53 protein Varied: dependent cells, VP22 has been found to spread to 100% on tumour origin of cells in a monolayer culture. Interestingly, T- and B-cell immunodeficiencies V(D)J recombination Lymphoid these intercellular trafficking properties also activating protein progenitor cells apply to VP22 fusion proteins. Because the Adenosine deaminase-induced Adenosine deaminase T cells, bone delivery of therapeutic genes or gene prodsevere combined marrow stem cells ucts into sufficient numbers of tumour cells immunodeficiency (SCID/ADA) remains a major difficulty, the use of VP22 fusion proteins could significantly increase the percentage of targeted cells. Furthermore, a recent study by possibly owing to the bacterial CDA protein functioning as a superWybranietz et al.31 has demonstrated that spread of VP22 fusion pro- antigen, resulting in polyclonal activation of T-lymphocytes. Subteins is a general biological phenomenon that is not restricted to dis- sequent studies using tumour cell lines that produced both CDA and tinct tissues or species. In principle, this approach could easily be IL-6 showed a failure to establish tumours upon subcutaneous injecadapted for potentiation of the bystander effect in solid tumours by tion in immunocompetent mice. It was thought that T cells and natural generation of VP22–HSV-tk or VP22–CDA fusion genes. killer (NK) cells were responsible for a majority of the activity and The use of interleukin 2 (IL-2) as an adjunctive therapy to HSV-tk that IL-6 might have been enhancing the T-cell response or antigen expression has been explored by Chen et al.39 Tumours were pro- presentation by B cells. duced in BALB/c mice by intraperitoneal injection of a colon cancer Santodonato et al.42 have combined the use of vaccination and cell line (MCA26). HSV-tk and IL-2 were delivered using two adeno- gene therapy with HSV-tk. Metastatic Friend erythroleukaemia cell viral vectors. GCV treatment resulted in large-scale tumour necrosis, (FLC) tumours are suppressed following vaccination with interferon with three out of the ten animals tumour-free. Significantly, an ap- a (IFN-a)-producing FLCs. Using IFN-a-producing FLC lines that preciable CTL response against the MCA26 cells was seen only in have been transfected with the gene for HSV-tk, a 70–100% cure rate those mice receiving both HSV-tk and IL-2. A further study using can be demonstrated in mice, with no secondary tumour formation. combined HSV-tk–IL-2 therapy in a murine oral cancer model, These mice are resistant to subsequent challenge with the wild-type showed a median survival time of three weeks compared with two parental tumour cells. High-dose chemotherapy with autologous BMT is an important weeks in mice that received HSV-tk alone, and one week in those treatment for patients with breast cancer. Purging techniques are rewith lacZ as a control gene40. Mullen et al.41, using the CDA–5-FC suicide system, transfected quired to limit potential contamination of the autologous graft by weakly immunogenic C57BL/6-derived colon cancer and fibrosar- residual tumour cells. Selective targeting of these tumour cells with coma cell lines and subcutaneously injected these cells into syn- a suicide-gene-containing vector offers a promising approach. Feasigeneic mice. Treatment with 5-FC resulted in substantial tumour bility tests in various mouse transplantation models are currently regression. Significantly, a reduction in secondary tumour formation under way. In one, Nanakorn et al.43, using adenoviral-vector–CDA upon challenge with syngeneic wild-type tumour cells was noted, gene targeting of breast cancer cells (BCCs) in mixtures of BCCs and
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haematopoietic cells, have shown sustained engraftment after 14 days of 5-FC treatment post-transplant. They now propose that peripheral blood stem cells from breast cancer patients undergoing autografts should be exposed in vitro to the adenoviral vector, followed by transplantation and a 14-day 5-FC treatment for in vivo purging of BCCs.
Human studies and clinical trials Ethical approval was given for the first gene therapy clinical trial as recently as 1990 and over 300 more such trials have been initiated. Although a majority of these trials are in Phase I/II stage, it is indicative of the rate at which this field is progressing and the urgency for this therapy. Over half of these trials involve therapeutic approaches to malignant disorders with approximately 40 for the correction of monogenic diseases such as severe combined immunodeficiency–adenosine deaminase deficiency (SCID–ADA), cystic fibrosis and a1-antitrypsin deficiency. The full scope is even wider, covering disorders that range from atherosclerosis to arthritis and Duchenne muscular dystrophy. Suicide genes have been used both as a safety mechanism secondary to the therapeutic gene and as the primary therapeutic gene indicating their importance and versatility in gene therapy. Lack of space does not allow a detailed account of these trials, but a few examples are offered. The first clinical trial to use a suicide gene as the primary therapeutic agent was approved in 1991 (Ref. 44) for the treatment of ovarian carcinoma. Ovarian cancer cells were transfected ex vivo with the gene for HSV-tk and then instilled intraperitoneally in nine patients in stage III of the disease. One patient achieved complete remission, whereas others showed partial tumour regression. It is arguable that use of more efficient methods of delivery that have been developed since would lead to better results. BMT is a potentially curative procedure in selected patients with haematological malignancies. Graft–versus–host disease (GVHD) remains a major problem following non-T-cell-depleted allogeneic BMT. Although donor T cells play a central role in both the graft–versus–leukaemia effect and immune reconstitution, the individual risk for GVHD cannot be predicted; ex-vivo transduction of these cells with a suicide gene before BMT offers a promising means for eliminating them at will. This is an approach that is being followed, for example with the truncated form of the low-affinity nerve growth factor gene (DLNGRF)–CDA–5-FC suicide system in a paediatric cohort. Bordignon et al.45, using a bicistronic retroviral vector, have transferred the HSV-tk gene and DLNGRF gene into allogeneic donor PBLs. The expression product of the DLNGRF gene was used as a marker for selection and enumeration of transduced cells. A total of 12 patients who suffered from severe complications following alloBMT or immunodeficiency were enrolled. A follow-up report has been published on eight of these patients46 revealing that three developed GVHD. Treatment with GCV resulted in elimination of transduced lymphocytes and GVHD, confirming the therapeutic potential of suicide gene therapy in the context of allo-BMT and GVHD. Several trials are under way for the treatment of malignant gliomas using the suicide gene approach and early results are
indicative of anti-tumour responses in several patients47. The brain deserves a special mention as an example of a tissue with relatively low immunological background, and low level of cell division. These features must be considered when devising strategies for gene therapy in this organ. An inherent advantage is that long-term gene expression is not required to effect cell death. Furthermore, the use of retroviral vectors provides selective delivery of the suicide gene to actively dividing tumour cells. Delivery can be enhanced further by cytokine stimulation of tumour cell growth. Results from these trials are awaited with anticipation.
Concluding remarks The need for gene therapy is paramount and the role of suicide genes as primary therapeutic agents and as safety switches in the delivery of other genes has been established. Although encouraging results are emerging from pre-clinical and clinical studies, the need for more efficient and specific methods of delivery are constantly being highlighted. Promising new avenues of research point to the eventual dovetailing of suicide gene therapy with more traditional therapies, resulting in novel techniques for patient care (Box 1). The dismal prognosis in many disorders acts as a spur in finding a solution to these problems. Opportunities for tackling existing and emerging diseases through gene manipulation abound. We are optimistic that, in the not too distant future, suicide gene therapy will complement, and even supercede some of the more conventional modalities. Indeed, the art of suicide gene therapy will itself become a convention.
We thank J. Wessels for help in preparing the diagrams and M. Eyrich for comments and suggestions. S.L. is the recipient of a Graduate Student Fellowship from the fortüne Foundation of the University Clinic of Tübingen. We acknowledge support from the fortüne Program (grants #375/1996 and #438/1997), the Deutsche Forschungsgemeinschaft (grants SFB510-C4 and La649/11-1), the Federal Ministry of Education, Science, Research and Technology (grant Fö. 01KS9602), the Interdisciplinary Research Centre (IKFZ) Tübingen and the Deutsche José Carreras Leukaemie-Stiftung (grant #DJCLS-R21).
Shangara Lal ([email protected]), Dietrich Niethammer and Paul Schlegel ([email protected]) are at the Dept of Paediatric Haematology and Oncology, Children’s Hospital, University of Tübingen, D-72076 Tübingen, Germany; Ulrich Lauer is at the Dept of Internal Medicine, University of Tübingen, D-72076 Tübingen, Germany; James Beck is at the University Children’s Hospital, Ernst-Moritz-Arndt University of Greifswald, D-17487 Greifswald, Germany.
References 1 Russel, S.J. (1993) Gene therapy for cancer. Cancer J. 6, 21–25 2 Davis, B.M. et al. (1996) Current progress in the gene therapy of cancer. Curr. Opin. Oncol. 8, 499–508 3 Verma, I.M. and Somia, N. (1997) Gene therapy – promises, problems and prospects. Nature 389, 239–242
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