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CONFERENCE

Gene delivery andtherapy strategies

Retroviruses have been the most commonlyused vectors in gene-therapy studies sincemuch is known about their structure andfunction. However, adenoviruses are show-ing promise as a gene vector because of theease with which they can be produced in the ’,concentrations necessary for efficient in-vivo gene-transfer. They formed one of thethree newer gene transfer systems pre-sented at the New York Academy ofSciences conference on Gene Therapy forNeoplastic Diseases held last month. :

Steven Brody (National Heart, Lung andBlood Institute, USA) discussed the use ofthree adenovirus-based vectors to transfera marker gene (P-galactosidase) or thetherapeutic genes al-antitrypsin and cysticfibrosis transmembrane conductance regu-lator (CFTR) cDNA. In-vitro and in-vivodata show that several tissues are highlyreceptive for adenovirus-mediated genetransfer. A clinical trial of CFTR cDNA :delivered down the trachea has begun at theUS National Institutes of Health. :

Another means of gene transfer is basedon the receptor-mediated endocytosistransport system.1 David Curiel (Univer-sity of Alabama, Birmingham, USA)described how biological molecules, actingas ligands, bind to specific cell-surfacereceptors and use this process to becomeinternalised. Curiel has constructed highlyefficient gene-transfer vehicles that containforeign genes and that mimic biologicalmolecules. Using transferrin as a modelligand, he has shown that foreign genes canbe internalised and expressed in cells pos-sessing transferrin receptors. Targeting oftumour cells by using lectins as ligands is apossibility since they can bind to tumour-cell-surface glycoproteins. :Leaf Huang (University of Pittsburgh,

Pennsylvania, USA) described the use of =

liposomes as a vehicle for genes. One of themajor problems of liposome-mediated genetransfer is toxicity caused by the lipidcomponent of the DNA-liposome complexto target cells. Toxicity can be reduced bychanging the lipid content of liposomes,but at the expense of stability. Liposomesknown as DC-Chol give the best ratiobetween toxicity and stability,2 and arebeing used in a clinical trial for melanoma(see below). Huang also presented data onan elegant cytoplasmic expression systemwhereby marker genes, under the control ofthe bacteriophage T7 promoter, were co-delivered with T7 RNA polymerase to vtarget cells with DC-Chol liposomes.3The introduction of suicide genes and

induction of drug sensitivity in tumourcells was discussed by Brian Huber (Well-come Research Laboratories, North Caro-lina, USA) and Karol Sikora (RoyalPostgraduate Medical School, London,

UK). The two studies presented are basedon the gene-therapy strategy known asVDEPT (virus directed enzyme prodrugtherapy), which uses a retroviral vector todeliver a foreign gene into a cell. The

foreign gene encodes an enzyme that con-verts a harmless prodrug into a cytotoxiccompound. Cytosine deaminase, for ex-ample, converts the relatively harmless5-fluorocytosine into the cytotoxic 5-

fluorouracil. The viral vector is capable ofinfecting both normal and cancer cells.

Expression of the enzyme gene is limited totumour cells by linking the gene to a

molecular switch that will be turned on

only in cancer cells. Sikora’s group createda "chimeric minigene" in which the en-zyme gene is linked downstream to part ofthe erbB2 promoter; increased activity ofthis promoter has recently been reported toproduce erbB2 overexpression in breastcancer.3 When the chimeric minigene wasdelivered into erbB2-positive and erbB2- :negative cells, significant expression of theprodrug activating gene cytosine dea-minase was highly expressed only in tu-mour cells overexpressing erbB2. When theprodrug 5-fluorocytosine was given tothese cells, cell death was observed. Moreimportantly, there was no prodrug activa-tion when 5-FC was given to erbB2 non-expressing cells that contained the chimericgene, thus demonstrating tumour-

specificity. Sikora’s group is using pancrea-tic and breast cancer models. :Michael Blaese (National Cancer Insti-

tute, NIH, USA) discussed the "bystandereffect" often seen in experiments involvingprodrug activating enzymes such as herpessimplex virus thymidine kinase (HSV-tk),which phosphorylates ganciclovir to toxicmetabolites. In a mixture of tumour cells inwhich only half express HSV-tk, over 90%of the cells in the mixture die upon ex-

posure to prodrug. He suggested that thiseffect may be due to the transfer of toxicmetabolites through tumour cell gap junc-tions. This effect could well be used tocircumvent the impossibility of targetingevery single cancer cell in vivo. :Immunomodulation of tumour cells to

invoke an immune response was another

: stimulating area for discussion. Cellular

and humoral immune effectors, such asMHC-restricted cytotoxic T cells, inhibitthe growth of cancer cells. The cytotoxic Tcells recognise MHC antigens on the

tumour cell surface and elicit an immune

response. The deficient expression of

MHC class I molecules on some tumour

cells is thought to contribute to their

evasion of the immune system. Gary Nabel(University of Michigan, Ann Arbor,USA) presented in-vitro and in-vivo datathat indicated gene transfer of an allogeneicMHC class I gene into melanoma cells.4

The expression of the foreign MHC anti-gen on the surface of transduced tumourcells induced a cytotoxic T-cell responseand resulted in partial tumour regression(as a consequence of enhanced tumourimmunogenicity). Interestingly, the ex-

pression of the foreign MHC gene in

tumour cells resulted in a specific cytotoxicT-cell response against previously un-recognised antigens on unmodified tumour

cells. A recently initiated clinical trial basedon these findings is the first in which genesare delivered directly into the tumourcell-a DC-Chol liposome preparationcontaining HLA-B7 genes is injected intocutaneous melanomas in HLA-B7-

negative patients to assess the toxicity ofdifferent doses of injected DNA.The use of genetically engineered

tumour-infiltrating lymphocytes (TILs)for the adoptive immunotherapy of me-lanoma was discussed by Patrick Hwu

(National Cancer Institute, NIH). 9

patients have been treated with TILs

expressing tumour necrosis factor (TNF)in a phase I dose-escalation toxicity study.Considerable controversy still surroundsthis approach since the concentration ofTNF released may not be sufficient for a

therapeutic effect.In only four years, over 25 trials of gene

therapy have been initiated in cancer

patients. It is too early to say whether thesewill be successful, but the conference gave a

glimpse of what lies ahead.

Jonathan Harris

1 Curiel DT. Adenovirus facilitation ofmolecular conjugate-mediated gene transfer.Prog Med Virol 1993; 40: 1-18.

2 Farhood H, Bottega R, Epand RM, et al.Effect of cationic cholesterol derivatives on

gene transfer and protein. Biochim BiophysActa 1992; 1111: 239-46.

3 Gao X, Huang L. Cytoplasmic expression ofa reporter gene by a co-delivery of T7 RNApolymerase and T7 promoter sequence withcationic liposomes. Nucl Acids Res 1993; 21:2867-72.

4 Hollywood DP, Hurst HC. A noveltranscription factor, OB2-1, is required foroverexpression of the proto-oncogene c-erbB-2 in mammary tumour lines. EMBO J1993; 12: 2369-75.

5 Plautz GE, Yang Z-Y, Wu B-Y, et al.Immunotherapy of malignancy by in vivogene transfer into tumours. Proc Natl AcadSci USA 1993; 90: 4645-49.