Peter F. Searle

Viral gene therapy strategies: from basic science to clinical application

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non‐toxic gene delivery system. Having evolved to deliver their genes to target cells, viruses are currently the most effective means of gene delivery and can be manipulated to express therapeutic genes or to replicate specifically in certain cells. Gene therapy is being developed for a range of diseases including inherited monogenic disorders and cardiovascular disease, but it is in the treatment of cancer that this approach has been most evident, resulting in the recent licensing of a gene therapy for the routine treatment of head and neck cancer in China. A variety of virus vectors have been employed to deliver genes to cells to provide either transient (eg adenovirus, vaccinia virus) or permanent (eg retrovirus, adeno‐associated virus) transgene expression and each approach has its own advantages and disadvantages. Paramount is the safety of these virus vectors and a greater understanding of the virus–host interaction is key to optimizing the use of these vectors for routine clinical use. Recent developments in the modification of the virus coat allow more targeted approaches and herald the advent of systemic delivery of therapeutic viruses. In the context of cancer, the ability of attenuated viruses to replicate specifically in tumour cells has already yielded some impressive results in clinical trials and bodes well for the future of this approach, particularly when combined with more traditional anti‐cancer therapies. Copyright

Vectors based on reducible polycations facilitate intracellular release of nucleic acids.

Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids.

Virus-Directed Enzyme Prodrug Therapy: Intratumoral Administration of a Replication-Deficient Adenovirus Encoding Nitroreductase to Patients With Resectable Liver Cancer

PURPOSE Virus-directed enzyme prodrug therapy depends on selective delivery of virus encoding a prodrug-activating enzyme to tumor, followed by systemic treatment with prodrug to achieve high levels of the activated cytotoxic at the intended site of action. The use of the bacterial enzyme nitroreductase to activate CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a short lived, highly toxic DNA cross-linking agent has been demonstrated in tumor xenografts. In this study, we report the first clinical trial investigating the feasibility, safety, and transgene expression of a replication-defective adenovirus encoding nitroreductase (CTL102) in patients with liver tumors. PATIENTS AND METHODS Patients with resectable primary or secondary (colorectal) liver cancer received a single dose of CTL102 delivered by direct intratumoral inoculation 3 to 8 days before surgical resection. RESULTS Eighteen patients were treated with escalating doses of CTL102 (range, 10(8)-5 x 10(11) virus particles). The vector was well tolerated with minimal side effects, had a short half-life in the circulation, and stimulated a robust antibody response. Dose-related increases in tumoral nitroreductase expression measured by immunohistochemical analysis have been observed. CONCLUSION Direct intratumoral inoculation of CTL102 to patients with primary and secondary liver cancer is feasible and well tolerated. The high level of nitroreductase expression observed at 1 to 5 x 10(11) virus particles mandates further studies in patients with inoperable tumors who will receive CTL102 and CB1954.

Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme.

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant ∼10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.

Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954

Expression of the E. coli enzyme nitroreductase (NTR) in tumour cells enables them to activate the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide), leading to inter- strand DNA crosslinking and cell death. Using transfected or retrovirally transduced SKOV3 ovarian carcinoma cell clones, we show a strong correlation between sensitivity to CB1954 and level of NTR enzyme activity. Importantly for clinical application in ovarian cancer, a cisplatin-resistant ovarian tumour cell line remains as susceptible to the NTR-dependent cytotoxicity of CB1954 as parental cells. In mixed populations of NTR-expressing and non-expressing cells, we observe a marked ‘bystander killing’ effect with this system. The use of NTR-encoding retroviruses from clonal producer cell lines at titres of 5 × 105 c.f.u./ml to transduce either established or low passage primary ovarian carcinoma lines only achieves an average 10-fold sensitisation of the cultures at gene transfer efficiencies of 15–25%. Concentration of the retrovirus to 3 × 107 c.f.u./ml elevates gene transfer to 80–90% in a single exposure to target cells, resulting in up to 500-fold sensitisation of the entire, unselected SKOV3 population to CB1954. In an initial investigation of NTR/CB1954 for the treatment of tumours in vivo, we observe regression of tumours expressing NTR following administration of CB1954, resulting in significantly increased median survival.

In vivo gene therapy for colon cancer using adenovirus-mediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase.

Virus-directed enzyme prodrug therapy (VDEPT) utilising cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) into the chemotherapy agent, 5-fluorouracil (5-FU), and has entered into a clinical trial for metastatic colon cancer. To improve this system, a replication-deficient adenovirus, containing a bifunctional fusion gene, CD:uracil phosphoribosyltransferase (UPRT), was constructed (AdCDUPRT). UPRT enhances the conversion of 5-FU into its active metabolites, which inhibit DNA and RNA synthesis. In vitro, AdCDUPRT infection of colon cancer cells resulted in a marked increase in sensitisation to 5-FU, compared with AdCD-infected or uninfected cells. The corollary is a ∼100-fold and ∼10 000-fold increase in sensitisation to 5-FC in AdCDUPRT-infected cells, compared to AdCD-infected and uninfected cells, respectively. There was a strong bystander effect in vitro, 70% of tumour cells were killed by 5-FC when only 10% of cells expressed CDUPRT. In vivo, athymic mice with colon cancer xenografts treated with intratumoral AdCDUPRT and intraperitoneal 5-FC, significantly reduced tumour growth rates compared with untreated controls (P = 0.02), whereas AdCD/5-FC treated mice did not. At higher AdCDUPRT virus doses, 5-FC and 5-FU were equally effective at delaying tumour growth compared with controls. In summary, VDEPT for colon cancer utilising AdCDUPRT is more effective than AdCD and the bifunctional CDUPRT gene enables the use of either 5-FC or 5-FU as prodrugs.

A Phase I/II Clinical Trial in Localized Prostate Cancer of an Adenovirus Expressing Nitroreductase with CB1984

We report a phase I/II clinical trial in prostate cancer (PCa) using direct intraprostatic injection of a replication defective adenovirus vector (CTL102) encoding bacterial nitroreductase (NTR) in conjunction with systemic prodrug CB1954. One group of patients with localized PCa scheduled for radical prostatectomy received virus alone, prior to surgery, in a dose escalation to establish safety, tolerability, and NTR expression. A second group with local failure following primary treatment received virus plus prodrug to establish safety and tolerability. Based on acceptable safety data and indications of prostate-specific antigen (PSA) responses, an extended cohort received virus at a single dose level plus prodrug. The vector was well tolerated with minimal side effects, had a short half-life in the circulation, and stimulated a robust antibody response. Immunohistochemistry of resected prostate demonstrated NTR staining in tumor and glandular epithelium at all dose levels [5 × 1010-1 × 1012 virus particles (vp)]. A total of 19 patients received virus plus prodrug and 14 of these had a repeat treatment; minimal toxicity was observed and there was preliminary evidence of change in PSA kinetics, with an increase in the time to 10% PSA progression in 6 out of 18 patients at 6 months.We report a phase I/II clinical trial in prostate cancer (PCa) using direct intraprostatic injection of a replication defective adenovirus vector (CTL102) encoding bacterial nitroreductase (NTR) in conjunction with systemic prodrug CB1954. One group of patients with localized PCa scheduled for radical prostatectomy received virus alone, prior to surgery, in a dose escalation to establish safety, tolerability, and NTR expression. A second group with local failure following primary treatment received virus plus prodrug to establish safety and tolerability. Based on acceptable safety data and indications of prostate-specific antigen (PSA) responses, an extended cohort received virus at a single dose level plus prodrug. The vector was well tolerated with minimal side effects, had a short half-life in the circulation, and stimulated a robust antibody response. Immunohistochemistry of resected prostate demonstrated NTR staining in tumor and glandular epithelium at all dose levels [5 x 10(10)-1 x 10(12) virus particles (vp)]. A total of 19 patients received virus plus prodrug and 14 of these had a repeat treatment; minimal toxicity was observed and there was preliminary evidence of change in PSA kinetics, with an increase in the time to 10% PSA progression in 6 out of 18 patients at 6 months.

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5‐(aziridin‐1‐yl)‐2,4‐dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a “suicide enzyme” for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication‐defective adenovirus vector (Ad‐CMV‐ntr), which allowed efficient gene transfer to SK‐OV‐3 or IGROV‐1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2,000‐fold sensitisation) with 25–50 p.f.u. per cell. Similar Ad‐CMV‐ntr‐dependent sensitisation to CB1954 was seen in 3 of 6 low‐passage primary ovarian tumour lines. Cells grown at low‐serum concentration to inhibit proliferation remained equally susceptible to the Ad‐CMV‐ntr‐dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme‐prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin‐resistant cells were sensitised towards CB1954 by Ad‐CMV‐ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin‐resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad‐CMV‐ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001). Int. J. Cancer 86:848–854, 2000.

NITROREDUCTASE: A PRODRUG‐ACTIVATING ENZYME FOR CANCER GENE THERAPY

1. The prodrug CB1954 (5‐(aziridin‐1‐yl)‐2,4‐dinitrobenzamide) is activated by Escherichia coli nitroreductase (NTR) to a potent DNA‐crosslinking agent.

Expression of Escherichia coli B nitroreductase in established human tumor xenografts in mice results in potent antitumoral and bystander effects upon systemic administration of the prodrug CB1954

Expression of the Escherichia coli enzyme nitroreductase (NTR) in mammalian cells enables them to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), leading to interstrand DNA cross-linking and apoptosis in both proliferating and quiescent cells. In the work reported here, we used human hepatocellular carcinoma and squamous carcinoma cell lines constitutively expressing NTR to demonstrate that the ntr/CB1954 system results in potent, long-lasting antitumoral effects in mice. We also demonstrate that this enzyme/prodrug combination results in antitumoral effects in vivo when only a minority of tumor cells express the enzyme, using either cells constitutively expressing NTR or ntr gene delivery in situ.