Clostridial spores as live 'Trojan horse' vectors for cancer gene therapy: comparison with viral delivery systems

Solid tumours account for 90% of all cancers. Gene therapy represents a potential new modality for their treatment. Up to now, several approaches have been developed, but the most efficient ones are the viral vector based gene therapy systems. However, viral vectors suffer from several deficiencies: firstly most vectors currently in use require intratumoural injection to elicit an effect. This is far from ideal as many tumours are inaccessible and many may have already spread to other parts of the body, making them difficult to locate and inject gene therapy vectors into. Second, because of cell heterogeneity within a given cancer, the vectors do not efficiently enter and kill every cancer cell. Third, hypoxia, a prevalent characteristic feature of most solid tumours, reduces the ability of the viral vectors to function and decreases viral gene expression and production. Consequently, a proportion of the tumour is left unaffected, from which tumour regrowth occurs. Thus, cancer gene therapy has yet to realise its full potential. The facultative or obligate anaerobic bacteria have been shown to selectively colonise and regerminate in solid tumours when delivered systemically. Among them, the clostridial spores were easy to produce, stable to store and safe to use as well as having extensive oncolytic ability. However, research in animals and humans has shown that oncolysis was almost always interrupted sharply at the outer rim of the viable tumour tissue where the blood supply was sufficient. These clostridial spores, though, could serve as "Trojan horse" for cancer gene therapy. Indeed, various spores harbouring genes for cancerstatic factors, prodrug enzymes, or proteins or cytokines had endowed with additional tumour-killing capability. Furthermore, combination of these "Trojan horses" with conventional chemotherapy or radiation therapies often significantly perform better, resulting in the "cure" of solid tumours in a high percentage of animals. It is, thus, not too difficult to predict the potential outcomes for the use of clostridial spores as "Trojan horse" vectors for oncolytic therapy when compared with viral vector-mediated cancer therapy for it be replication-deficient or competent. However, to move the "Trojan horse" to a clinic, though, additional requirements need to be satisfied (i) target tumours only and not anywhere else, and (ii) be able to completely kill primary tumours as well as metastases. Current technologies are in place to achieve these goals.     

The gene suicide system Ntr/CB1954 causes ablation of differentiated 3T3L1 adipocytes by apoptosis

The feasibility of ablating differentiated adipocytes and the mechanism of cell ablation with a suitable prodrug activating system is described. The system is based on the use of E. coli nitroreductase (NTR) enzyme that activates certain nitro compounds, such as the antitumor drug CB1954, into cytotoxic DNA interstrand cross-linking agents. Differentiated preadipocyte cells (3T3L1) transfected with an aP2 driven nitroreductase construct were efficiently killed after incubation with medium containing the prodrug CB1954, while untransfected cells were not affected. It was demonstrated that the mechanism of cell ablation is apoptosis and that the system has a bystander effect mediated by a toxic metabolite of the prodrug. The described system should provide a good alternative approach for gene therapy studies and a new inducible approach to manipulating the number of cells in tissues of transgenic animals and the ability to study the recovery of the tissue from cell damage or loss.     

Purine nucleoside phosphorylase and fludarabine phosphate gene-directed enzyme prodrug therapy suppresses primary tumour growth and pseudo-metastases in a mouse model of prostate cancer

Gene-directed enzyme prodrug therapy based on the E. coli purine nucleoside phosphorylase (PNP) gene produces efficient tumour cell killing. PNP converts adenosine analogs into toxic metabolites that diffuse across cell membranes to kill neighbouring untransduced cells (PNP-GDEPT). Interference with DNA, RNA and protein synthesis kills dividing and non-dividing cells, an important consideration for slow-growing prostate tumours. This study examined the impact of administering PNP-GDEPT into orthotopically grown RM1 prostate cancers (PCas) on the growth of lung pseudo-metastases of immunocompetent mice. C57BL/6 mice bearing orthotopic RM1 PCas received a single intraprostatic injection of OAdV220 (10(10) particles), a recombinant ovine atadenovirus containing the PNP gene controlled by the Rous Sarcoma virus promoter, followed by fludarabine phosphate (approximately 600 mg/m(2)/day) administered intraperitoneally (ip) once daily for 5 days. Pseudo-metastases were induced 2 days after intraprostatic vector administration by tail-vein injection of untransduced RM1 cells. Mice given PNP-GDEPT showed a significant reduction both in prostate volume (approximately 50%) and in lung colony counts (approximately 60%). Apoptosis was increased two-fold in GDEPT-treated prostates compared with controls (P < 0.01), but was absent in the lungs. Staining for proliferating cell nuclear antigen (PCNA) indicated that proliferation of both RM1 prostate tumours (P < 0.01) and lung colonies (P < 0.01) was significantly suppressed after GDEPT. Although prostate tumour immune cell infiltration did not differ significantly between treatments, immunostaining for Thy-1.2 (CD90) showed that GDEPT promoted Thy-1.2(+) cell infiltration into the prostate tumour site. This study showed that a single course of PNP-GDEPT significantly suppressed local PCa growth and reduced lung colony formation in the aggressive RM1 tumour model.     

Clostridia in cancer therapy

During the past decade, the search for an effective system for the selective delivery of high therapeutic doses of anti-cancer agents to tumours has explored a variety of ingenious and increasingly complex biological systems. These systems are most often based on gene therapy and use viral vectors as the delivery vehicle. Invariably, such systems have been found wanting with respect to a lack of tumour specificity, poor levels of transgene expression and inefficient distribution of the vector throughout the tumour mass. By contrast, the ability of intravenously injected clostridial spores to infiltrate, then selectively germinate in, the hypoxic regions of solid tumours seems to be a totally natural phenomenon, which requires no fundamental alterations and is exquisitely specific.     

The use of clostridial spores for cancer treatment

Hypoxic/necrotic regions, absent in normal tissues, can be exploited to target tumours in cancer therapy using nonpathogenic strains of the bacterial genus Clostridium. Following administration of Clostridium spores to tumour-bearing organisms, these spores can only germinate within the hypoxic/necrotic regions of solid tumours, proving their exquisite selectivity. Low oxygen tension is a common feature of solid tumours, which may arise from the unique physiological environment, generated to a large extent by the abnormal tumour vasculature, and provides as such a niche for anaerobic bacteria. Some clostridia tested clearly showed innate oncolytic activity, but they could not completely eradicate the tumour. Recombinant clostridia producing prodrug-converting enzymes or cytokines resulted in the production of such proteins solely within the tumour, and where applicable, could convert the prodrug in a toxic compound. Moreover, in some cases, tumour eradication or tumour control could be observed. This review brings an overview of the relative successes and failures of the Clostridium-directed tumour therapy with both wild-type strains and strains producing proteins useful in antitumour therapy.     

An unusually cold active nitroreductase for prodrug activations

A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.     

Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: synthesis,stability, enzymatic reduction,and antiproliferative activity in cell culture

In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C. However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E. coli nitroreductase-expressing cells. These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E. coli nitroreductase. Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.     

Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae

The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.     

Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.     

Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.     

Systems biology and the mathematical modelling of antibody-directed enzyme prodrug therapy (ADEPT)

Antibody-directed enzyme prodrug therapy (ADEPT) can generate highly localised concentrations of cytotoxic agents directly in a tumour, thereby reducing the collateral toxicity associated with normal tissue exposure. ADEPT is a two-component approach. First, a non-toxic antibody-enzyme fusion protein is localised in the tumour matrix by binding a specific antigen expressed only on the surface of a cancer cell. Once the fusion protein is bound, an inert small molecule prodrug is administered which is the substrate for the enzyme bound to the tumour surface. When the prodrug comes into contact with the bound enzyme, an active cytotoxic agent is generated. A multiple length-scale model of ADEPT therapy in solid tumours is presented. A four-compartment pharmacokinetic (PK) model is formulated where the tumour is comprised of interstitial and cell-surface subcompartments. The macroscopic PK model which describes the biodistribution of antibody-enzyme conjugate, prodrug and active drug at the largest length scale is coupled to a reaction-diffusion tumour model. The models are qualitatively validated against current literature and experimental understanding. The relationship between tumour localisation and the affinity of the antibody-enzyme conjugate for its surface antigen is explored by simulation. The influence of pharmacokinetic and biophysical parameters such as renal elimination rate and permeability of the tumour vasculature upon tumour uptake and retention of the fusion protein are also explored. Lastly, a technique for establishing an optimal prodrug dosing schedule is formulated and initial simulation results are presented.     

Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase

In order to generate a zebrafish model of beta cell regeneration, we have expressed an Escherichia coli gene called nfsB in the beta cells of embryonic zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme, which can convert prodrugs such as metronidazole (Met) to cytotoxins. By fusing nfsB to mCherry, we can simultaneously render beta cells susceptible to prodrug and visualize Met dependent cell ablation. We show that the neighboring alpha and delta cells are unaffected by prodrug treatment and that ablation is beta cell specific. Following drug removal and 36h of recovery, beta cells regenerate. Using ptf1a morphants, it is clear that this beta cell recovery occurs independently of the presence of the exocrine pancreas. Also, by using photoconvertible Kaede to cell lineage trace and BrdU incorporation to label proliferation, we investigate mechanisms for beta regeneration. Therefore, we have developed a unique resource for the study of beta cell regeneration in a living vertebrate organism, which will provide the opportunity to conduct large-scale screens for pharmacological and genetic modifiers of beta cell regeneration.     

The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli.

The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis.     

腺病毒载体在生物治疗中的应用

腺病毒载体是目前重要的基因转移载体之一。腺病毒可作为真核基因表达载体,可制成灭活、重组或抗癌疫苗用于预防呼吸道疾病、癌症和肝炎等传染病。在癌症的基因治疗方面,Ａｄ 载体可运载肿瘤抑制基因,自身基因编码蛋白能诱导细胞调亡,可作为前药物感染细胞,还能利用Ａｄ 的一些特殊复制子,达到治疗肿瘤的目的     

Regulation of alpha- and beta-secretase activity by oxysterols: cerebrosterol stimulates processing of APP via the alpha-secretase pathway

Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.     

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.     

Nitrobenzylcarbamate prodrugs of cytotoxic acridines for potential use with nitroreductase gene-directed enzyme prodrug therapy

The synthesis, solvolytic behaviour and cytotoxicity of novel 4-nitrobenzyl carbamates and carbonates derived from 3-amino-4-hydroxymethylacridine 1 are described. Compounds 2 and 6 are both substrates for Escherichia coli nitroreductase and the highly active lead structure 1 is liberated upon incubation of the two compounds in the presence of NTR and its cofactor NADH. Additionally, the cytostatic activity of 2 and 6 against human HT29 colon carcinoma cell lines is decreased 80-fold and 360-fold, respectively, indicating their suitability and potency as prodrugs for either gene-directed enzyme prodrug therapy or antibody-directed enzyme prodrug therapy.     

Nitrobenzyl carbamate prodrugs of enediynes for nitroreductase gene-directed enzyme prodrug therapy (GDEPT)

The synthesis and evaluation of the 4-nitrobenzylcarbamate enediyne 6 and related compounds as prodrugs activated by a nitroreductase enzyme (NTR) from E. coli B is described. Expression of NTR in three different cell lines gives increases in cytotoxicity of 21- to 135-fold for 6 (IC₅₀ values 13–24 nM in the NTR-expressing lines), indicating its potential as a prodrug for NTR-mediated Gene-Directed Enzyme Prodrug Therapy. The cytotoxicity of 6 and related enediynes is shown to be oxygen-dependent, especially in nucleotide excision repair-proficient cells, which might limit activity in hypoxic regions of tumours.