Gene therapy in gynaecological cancers

Current Obstetrics & Gynaecology (1998)8, 218-223 © 1998Harcourt Brace & Co. Ltd

Gynaecology

Gene therapy in gynaecological cancers

S. T. Kehoe and N. Acheson The concept o f therapeutic interventions involving genetic manipulation adds another dimension to the treatment of malignant disorders. Targeting various oncogenes (proto and suppressor), inducing cell death through the intracellular activation of drugs, influencing immune activity and reducing toxicity with standard modalities are all amenable to genetic strategies. Gene therapy in gynaecological cancers, though still in its infancy, has developed to the stage where preliminary trials on humans have commenced. This article is by no means comprehensive, but presents some of the basic concepts of gene therapy and some recent studies which encompass ma W of the principles involved in the translation of the therapy to humans.

intracellular compartment and induce the desired effect for a sufficient duration without an adverse impact on the host. Genetic manipulation can be performed in vivo or ex vivo. In vivo is the modification of cells whilst in the host whereas ex vivo involves the removal of cells, transfection by the vector carrying the message, and then re-introduction to the host.

INTRODUCTION The advances made in molecular biology have permitted the elucidation of cellular functions involved in tumourigenesis. Such knowledge enables the targeting of these abnormalities in a manner not compatable with standard radio/chemotherapeutic modalities. The original application of gene therapy in humans involved genetic marking of tumour infiltrating lymphocytes (TILs), which were then infused into cancer patients. This was not undertaken for therapeutic purposes, but to gain information to enhance adoptive immunotherapeutic treatments. The pace of progress is evident by the fact that this original research was peribrmed within the last decade.

DELIVERY SYSTEMS: THE V E C T O R S

The basic vectors employed are either viruses (transduction) or mechanical (transfection) procedures (Table 1) each with inherent advantages and disadvantages. Viral vectors

BASIC P R I N C I P L E S

Retroviruses

Gene therapy involves delivering a message which, through its action, eliminates malignant cells. To achieve delivery a vector, which is required to accommodate the information package, must reach the

Retroviruses are one of the commonest type of vector used, containing an inner core of RNA (nucleotide) with a protective coat of protein (capsid). In viral transduction the viral genome is modified to incorporate the passenger gene (message). The modified or recombinant gene is then transferred to the target cells. The advantage of these vectors is their ability to introduce passenger genes into the host genome, thus, inducing a degree of permanency which facilitates long-term expression. The

Scan T. Kehoe,Senior Lecturer,Hon. Consultant in

GynaecologicalOncology,NigelAcheson,ResearchFellowin GynaecologicalOncology,Department of Gynaecological Oncology,City Hospital Trust, DudleyRoad, Birmingham B17 9HH, UK Correspondence to: S. K.

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Table 1 Vectorsystems Transductionmethods Viruses Retroviruses Adenoviruses Vaccinia Paroviruses

Chemical

Transfectionmethods Physical

Calcium Phosphate Liposomes Gold/TungstenBeads

major disadvantages include: (1) the ability to accommodate only a small load (8 kilobases) of information; (2) they may be rendered inactive by serum complement; and (3) they require active DNA replication for efficient integration. The last is necessary as nuclear translation with retroviruses involves the additional steps of reverse transcription of vector RNA to DNA. As most cells within a malignant mass are quiescent, only a small proportion of cells are transfected. However, as shown later, this is less problematic than it would seem. Another concern is the potential inadvertent transfection of proliferating normal cells with induction of malignant transformation, although in studies to date this has not materialized. Adenoviruses These are non-enveloped icosahedral viruses containing a double-stranded DNA genome. Adenoviruses contain early genes - transcribed before DNA replication - and late genes transcribed after DNA replication. Transfection with adenoviruses are not limited to replicating cells and though advantageous, integration of the passenger gene into the host genome does not occur. The result is a lack of permanency, necessitating repeated treatments. As a consequence, there is an increased risk of host immune reactions with elimination of the vector. A second generation of adenoviral vectors has been developed with prolonged activity which elicits less immune reactions) 3 Many other vectors, such as paraviruses and certain vaccinia, are undergoing development. Further details on these and other advances are available in references. ~6

Electrical Bombardment Direct Tumour Inoculation

cell-membrane barrier. Because cell-membrane barrier resistance differs between tissue types, delivery is variable. As with adenoviral vectors, the lack of integration into the host genome results in a short duration of action. Another consideration is accessibility and, whilst such therapy is applicable to external tumours (such as skin), it has a limited role for internal diseases.

Direct DNA injection Pure closed circular plasma DNA or RNA is injected directly into the desired tissue. The method is simple, inexpensive, non-toxic and capable of carrying a large message. Presently, incorporation and expression of the genome is considered to last for too short a time for effective therapy. Although access is an issue for many tumours, advances in surgical techniques may permit greater use of this system in the future.

Liposomes Monocationic and polycationic lipids form liposomes through their spontaneous binding with polyanionic DNA (or RNA). Fusion with cell surfaces (negative charge) then occurs, with delivery of the specific passenger gene. Some of the DNA transferred will relocate in the cell nucleus and use the host's transcription to express the passenger gene. Transfer is effective for both proliferating and non-proliferating cells, and this system is presently undergoing evaluation in clinical trials.

THE TARGETS

Non-viral or transgenic infection

Oncogenes/tumour suppressor genes

Both physical and chemical methods are used to achieve non-viral gene transfer. Physical methods involve particle acceleration or electroporation. Chemical methods include the use of calcium phosphate, liposomes and molecular conjugates.

Oncogenes are promoters of cellular proliferation. In malignancy, abnormalities can be due to gene amplification, over-expression of the product or gene mutation. An inability to function normally results in increased cellular growth. Tumour suppressor genes act as negative growth controllers. Their function is to recognize abnormal DNA, arrest the cell cycle and permit reparation of any defect. If this is not achieved, cell apoptosis is induced. Dysfunctions of tumour suppressor genes are normally due to point mutations, and defects result in enhanced cellular proliferation and malignant transformation. Important oncogenes

Particle bombardment Plasmid DNA, containing the specific message, is coated onto a 1-3 g diameter gold or tungsten bead. These are then accelerated by an electrical or gaseous device and fired at the tissue, penetrating the

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Table 2 Oncogenesuppressor geneexpressionin gynaecological cancers

Ovarian cancer

Cervical cancer Endometrial cancer Vulval cancer

Oncogene

Expression rates

c-erbB-2 K-ras p53 c-myc erbB-2 p53 c-myc

1740% 2040% 30 70% 10-55% 2540% 10@% 50%

erbB-2 p53 c-myc p53

9-14% 20% 10% variable

eliminated. Indeed, even 10% cellular transfections afforded excellent responses. The term 'bystander effect' was applied to this finding. The precise explanation of the events involved in this process is unclear. In vivo and in vitro studies yielded a variety of hypotheses. These include a direct transfer of toxic GCV metabolites to adjacent cells through phagocytosis of apoptotic vesicles, enhancement of local immune reaction and release of cytokines. Continued research into the mechanism(s) of this effect may permit its use to enhance gene therapy or, indeed, yield another therapeutic option?

Studies in gynaecological cancers in gynaecological cancers include c-erbB-2 (also known as HER-2/neu), c-myc and the ras gene family. P53 is a well recognized suppressor gene, as is the more recently discovered BRCA1 gene. 7 The variability in the expression of these gene abnormalities in many cancers means that targeting a single gene will be limited to the subpopulation of relevant cells (Table 2).

Clinical trials using gene therapy in gynaecological cancers have only recently received approval. These mainly involve ovarian cancer (Table 3) as the containment of the disease and ease of access to the peritoneal compartment facilitates therapy.

OVARIAN CANCER

Suicide gene therapy

Suicide gene therapy

Delivery of a cytotoxic agent specifically to malignant cells would overcome many of the unwanted sequelae of present therapies. Suicide gene therapy incorporates this by modifying the target cells followed by the administration of a non-active drug (pro-drug). Only with modification, are cells capable of transforming the pro-drug into its active constituent and consequently induce cell death (suicide gene therapy). Prodrugs may enter normal cells, but as they are unmodified, no adverse events occur.

The efficacy of this treatment has been confirmed in animal models. Using an adenoviral vector, Behbakat et al., 9 exposed ovarian cancer cells to vectors expressing a reporter gene (lacZ) and to the same vector bearing the HS-tk gene (AdRSVtk). Tumours were then induced in immunodeficient mice by intraperitoneal and subcutaneous inoculation. Treatment with the vector followed by GCV resulted in a 10-20-fold lower subcutaneous tumour burden, without any intraperitoneal tumour developing, when compared to untreated mice. Applying similar techniques, Tong et al? ° reported comparable results, but also noted that viral dose and tumour burden influenced the response rates. Even with the advantage of the bystander effect, Tong et al.'s study would imply the need to modify therapy according to the clinical scenario.

Herpes simplex thymidine kinase gene Because of its popularity for use in cancer therapy, this vector deserves specific mention. First applied in treating brain tumours (in rats), the delivery of this gene conferred sensitivity to the antiherpes drug ganciclovir (GCV). On entering the herpes simplex thyrnidine kinase gene (HS-tk) infected cells GCV is converted to GCV-monophosphate and, subsequently, the active GCV-triphosphate. This inhibits DNA-polymerase by incorporating into the DNA strand, which results in chain termination. The model employed is unique, in that proliferation within the brain is confined to the tumour, with normal neuronal cells remaining quiescent. Therefore, inadvertent transfection of the latter does not pose difficulties.

The bystander effect A serendipitous finding relating to the above studies was that not all cells within a tumour required transfection to achieve total turnout destruction. Nontransfected cells close to the HS tk cells were also

Oncogenes/tumour suppressor genes Over-expression of c-erbB-2 (HER-2/neu) is a recognized poor prognostic factor in ovarian cancer. This proto-oncogene is closely related to epidermal growth factor (erbB-1). The product is a 185 kDa membrane glycoprotein which acts as a growth factor receptor. Deshane et al. '~ targeted this gene using an adenovirally directed antibody (anti-erbB-2-antibody) and achieved a significant reduction in intraperitoneal tumour burden. Furthermore, this group demonstrated the confinement of therapy to the intraperitoneal milieu without adverse effects to any outside organs. This is important as, although over-expression of c-erbB-2 is abnormal, it still has obligatory functions in normal cellular proliferation. Expanding this

Gene therapy in gynaecological cancers

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Table 3 Approved phase I gene therapy trials in ovarian cancer* Institution

Therapy

Vector

Target

Type

Berchuck et al, University of Durham USA Hwu et al. NIH, Bethesda USA

II-2 gene modified cancer cells Anti-CD3 stimulated PBLs transduced with chimeric T-cell receptor gene I.E HSK-tk

Lipid

Immunotherapy

MFG-Movy

Metastatic Ov. Ca. PBLs

Adenovirus

Ov. Ca.

Chemotherapy

Modified HSV-tk

Tetrovirus

PA-10v. Ca,

Chemotherapy

Vaccination with HER-2/neu expressing tumour cells and HSV--tk gene modified cells HSV-tk treatment of refractory or recurrent ovarian cancer Protection of haematopoietic cells during chemotherapy for ovarian cancer BRCAI retroviral g e n e therapy for ovarian cancer E1A gene therapy for EOC over-expressing HER-2/neu

Retrovirus

PA-10v. Ca,

Chemotherapy

Retrovirus

Ov. Ca.

Chemotherapy

Retrovirus

Haematopoietic

Drug resistance

Retrovirus

Or. Ca.

Tumour suppressor/antisense

Lipid

Metastatic Ov. Ca.

Tumour suppressor/antisense

Curiel et al. University of Alabama USA Freeman et al. University Medical Centre, New Orleans, USA Freeman et al. University Medical Centre, New Orleans, USA Link et al. Human Gene Therapy Institute De Moines USA Deissroth et al. Hammersmith, London, UK Holt et al. NIH, Bethesda USA Hortobagyi et al. Anderson Centre, USA

Immunotherapy

Ov. Ca: ovarian cancer EOC: epithelial ovarian cancer *Compiled from information in Reference 3 and 6 approach, Wu et al. ~2constructed a retrovirus to target erbB-2 containing the neomycin resistant gene and erbB-2 antisense fragment, and combined this with standard chemotherapy. N o t only did transfection reduce cell growth, but it also enhanced sensitivity to 5-flurouracil and cisplatinum. Such a dual approach is an attractive form of therapy for ovarian cancer. Others have targeted erbB-2 using differing bIocking factors and vectors. K1 mutant SV40 large T antigen and 5E1A genes act as suppressors to erbB-2. Impressive survival outcomes were obtained with liposome transfer of K1 gene (administered weekly) to mice inoculated with over-expressing ovarian cell lines. At 1 year from treatment, 40% of mice were alive. This compares to all untreated mice dying within 7 months. ~ 5E1A gene therapy (which acts at the transcriptional level), again using liposomes, has also proven successful. Untreated mice (inoculated ip with SKOV-3 cell line over-expressors of HER-2/neu) all died within 160 days, whereas 70% of the treated group survived for 1 year. TM

Tumour suppressor genes Mutations of p53 are prevalent in many cancers presenting an obvious target for therapy. Eliminating mutant p53 or introducing wild type (normal) p53 into affected cells are two possibilities. Santosa et al. .5 tested the ability of an adenoviral construct ( A d - C M V - p 5 3 ) to introduce wild type p53 into a specific ovarian cancer cell line containing mutant p53.

Infection rates were high, with transfection of wild type p53 causing growth inhibition more than 90% of infected cells. Skilling et al. .6 on the other hand used antisense to mutant p53 which also inhibited growth without an adverse effect on cells with normal p53. Mutations of BRCA1 gene and the hereditary nature of a small proportion of ovarian cancers is now established. BRCA1 is a nuclear phosphoprotein which acts as a suppressor gene. Shao et al. ~7investigated the role of BRCA1 in specifically developed breast cancer cell lines. This revealed that BRCA1 may play a critical role in cell apoptosis, and that mutations of this gene could, in part, explain the lack of apoptotic capability in certain breast and ovarian malignancies. Increasing the apoptotic threshold of BRCA1 could, in theory, prevent disease spread, although further research is warranted prior to pursuing this course of action.

Drug resistance Drug resistance limits the efficacy of many standard treatments. The major gene involved in this process is multiple drug resistant gene-1 (MDR-1). Located on chromosome 7, it encodes for P-glycoprotein, with the role of detecting and removing drugs as they pass the plasma membrane. MDR-1 could be used to protect normal cells from toxic side effects, as a target, or by its elimination, to overcome drug resistance. All these possibilities are undergoing investigation, but the main area of interest is in reducing drug toxicity

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(in particular, bone marrow) to permit dose intensification. Sone et al. ~8 recently described targeting MDR-1 in nude mice inoculated with MDRAD10 cell lines transduced with human macrophage colony stimulating factor (M-CSF) gene. The cell lines that were developed produced either low, intermediate or high levels of M-CSE The systemic injection of antiMDR-1 antibody (MRK17) significantly reduced the growth of subcutaneous tumours in ML-AD10 tumours (low M-CSF producers). Adding recombinant M-CSF promoted growth of the parental cells (AD10). In vivo experiments showed that mixing parental cells with M-CSF modified cells with exposure to MRK17, enhanced the cytotoxic effect on both cell lines, indicating the potential for combination gene therapy.

Vaeeina therapy Recently, Borysiewicz et al. 22provided evidence as to the role of vaccinia therapy in cervical cancer. Targeting E6 and E7 with recombinant vaccinia virus (TA-HPV), which expressed the E6 and 7 proteins of both HPV16 and HPV18, a phase 1 study on eight patients with advanced cervical carcinoma revealed that all mounted an immune response to the vaccinia, with three patients producing HPV-specific cytotoxic T cells. No untoward events were noted. As immune competence is diminished in advanced malignancy, a better immune response would be predicted if therapy was administered in earlier stage disease. Should future research confirm these findings, the place of vaccinia in treating, and possibly preventing malignancy becomes a real possibility. However, adverse long-term effects remain an obstacle.

Immune/eytokine therapy Though Sone et al.'s study did employ an immunological component, most attention in this area of gene therapy has focused on enhancing the cytoxicity of the host immune system. The majority of research involves the ex vivo modification of TILs. Fujita et al. ~9demonstrated how the retroviral transduction of the fyn gene into TILs augmented the T-cell receptor CD3 complex signal transduction in TILs obtained from six patients with ovarian cancer. The expression of the transduced gene was five times more than that of the endogenous fyn gene, and TIL cytolytic activity against autologous (but not allogenic) tumour cells was significantly enhanced. Many other examples of T-cell modification are reported in the literature, and some speculate that immunologicallybased gene therapy may eventually prove the best form of intervention.

CERVIX Both HPV16 and HPV18 are detected in the majority of cervical cancer cells and have a role in oncogenesis by interfering with p53. The E6 and E7 genes of HPV being particularly involved in this process. Adenoviral vectors carrying antisense RNA transcripts (Ad5CMV-PV16AS) for E6 and E7 genes of HPV16, reduce cell growth in specifically produced cell lines. The therapeutic efficacy is improved by the addition of an adenoviral p53 construct (AdCMV-p53), as corroborated by animal studies? ° Chen et al?' assessed the ability of three hammerhead ribosomes to cleave RNA transcripts from E6 and E7 genes of HPV 18. Targets were located at nucleotides (nt) 123, 309 and 671 of the viral transcript. Successful hybridization to the target site was achieved, with effective cleavage by each ribosome - the most effective noted to be nt309. Future therapies directed at HPV can be predicted, but potentially immunological strategies could precede such interventions.

POTENTIAL DIFFICULTIES WITH GENE

THERAPY Although results are impressive, the models used were selected for their over-expression of the target gene. This is not the case in human cancers. Even within a single tumour, gene expression can vary between primary and metastatic disease, as shown by Provencher et al. 23 in relation to p53 and ovarian cancer. Therefore, multiple targeting may be required to ensure sufficient cell destruction. However, as reported by Janick et al.,24 this may pose problems. Their study involved targeting both c-myc (a proto-oncogene) and p53. Three cell lines were used (CAOV3, SKOV3 and BG1), with proliferation measured by ATP viability assays, after 6 days of treatment. Antisense therapy to the target regions were 27-mer antisense phosphorotioate oligodeoxyribonucleotides targeting the Puffnm23 binding region of cmyc and promoter/ATG region of p53. The effect varied between cell lines. Whilst growth of BG1 was inhibited at high doses, intermediate doses were associated with enhanced proliferation. Combination therapy was synergistic in CAOV3, but antagonistic in SKOV3. The unpredictable responses are of concern, especially if tumour growth is enhanced under certain conditions. CONCLUSIONS Developments in gene therapy can be anticipated to occur in parallel with new discoveries in molecular biology. Even with a somewhat limited understanding, the diversity of its potential is evident. Improvements in vectors and more specific cell targeting can be expected in the future. The only major doubts relate to unforeseen adverse effects. It is unrealistic to expect the results from clinical trials to achieve equal effects as shown in the laboratory setting. However, the era of gene therapy is a welcome addition to the treatment of malignant diseases.

Gene therapy in gynaecological cancers REFERENCES

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