Cancer Gene Therapy is the essential gene. Tumor-targeting prodrug-activating bacteria for cancer. we developed a strain of bacteria to express enzymes for selective prodrug activation and non-invasive imaging in. Directed enzyme prodrug therapy (DEPT) uses enzymes artificially introduced into the body to convert Prodrugs, which have no or poor biological activity, to the active form in the desired location within the body. Many.
Author Information. 1. Molecular Neurogenetics Unit, Massachusetts General Hospital, Department of Neurology, and Neuroscience Program, Harvard Medical School, Boston, MA 02114, USA. 2. Department of Neurology, Massachusetts.
Prodrug activation enzymes in cancer gene therapy - Aghi - 2. The Journal of Gene Medicine. Abstract. Among the broad array of genes that have been evaluated for tumor therapy, those encoding prodrug activation enzymes are especially appealing as they directly complement ongoing clinical chemotherapeutic regimes.
These enzymes can activate prodrugs that have low inherent toxicity using both bacterial and yeast enzymes, or enhance prodrug activation by mammalian enzymes. The general advantage of the former is the large therapeutic index that can be achieved, and of the latter, the non- immunogenicity (supporting longer periods of prodrug activation) and the fact that the prodrugs will continue to have some efficacy after transgene expression is extinguished. This review article describes 1. Essentially all of these prodrug activation enzymes mediate toxicity through disruption of DNA replication, which occurs at differentially high rates in tumor cells compared with most normal cells.
Publication » Nitroreductase: A prodrug-activating enzyme for. A prodrug-activating enzyme for cancer gene. nitroreductases were shown to be involved in high-value projects such as prodrug activation in gene. Prodrug activation enzymes in cancer gene therapy Aghi, Manish; Hochberg, Fred; Breakefield, Xandra O. Among the broad array of genes that have been evaluated for tumor therapy, those encoding prodrug activation enzymes are.
- Cancer Gene Therapy is the essential gene therapy resource for cancer researchers and clinicians, keeping readers up to date with the latest developments in gene therapy for cancer.
- The selective activation of prodrug in tumor tissues by exogenous enzyme for. including gene-directed enzyme prodrug therapy, virus-directed. The central part of enzyme/prodrug cancer therapy is to deliver drug.
In cancer gene therapy, vectors target delivery of therapeutic genes to tumor cells, in contrast to the use of antibodies in antibody- directed prodrug therapy. Vector targeting is usually effected by direct injection into the tumor mass or surrounding tissues, but the efficiency of gene delivery is usually low.
Nitroreductase: a prodrug-activating enzyme for cancer gene therapy. Searle PF(1), Chen MJ, Hu L, Race PR. which has identified a number of mutants with improved kinetics of CB1954 activation. Aghi M(1), Hochberg F, Breakefield XO. Author information: (1)Massachusetts General Hospital, Department of Neurology, Harvard Medical School, Boston 02114, USA. Among the broad array of genes that have been evaluated for. Use of substrates for human enzymes may allow prodrug activation in. Rehemtulla A. Toward an enzyme/prodrug strategy for cancer gene therapy: endogenous activation of carboxypeptidase A mutants by the PACE/furin.
Thus it is important that the activated drug is able to act on non- transduced tumor cells. This bystander effect may require cell- to- cell contact or be mediated by facilitated diffusion or extracellular activation to target neighboring tumor cells. Effects at distant sites are believed to be mediated by the immune system, which can be mobilized to recognize tumor antigens by prodrug- activated gene therapy.
Prodrug activation schemes can be combined with each other and with other treatments, such as radiation, in a synergistic manner. Use of prodrug wafers for intratumoral drug activation and selective permeabilization of the tumor vasculature to prodrugs and vectors should further increase the value of this new therapeutic modality. Copyright © 2. 00. John Wiley & Sons, Ltd.
Cancer Gene Therapy - Membrane- localized activation of glucuronide prodrugs by [beta]- glucuronidase enzymes. Cancer Gene Therapy (2. September 2. 00. 6K- C Chen.
T- L Cheng. 2,3, Y- L Leu. Z M Prijovich. 1, C- H Chuang. B- M Chen. 1 and S R Roffler. Top of page. Introduction.
Although many anti- neoplastic agents have demonstrated utility in the clinic, drug- induced toxicity to non- cancerous tissues can prevent attainment of therapeutic drug concentrations at cancer cells and lead to premature termination of therapy before achieving complete remission. More selective anti- neoplastic agents could positively influence therapy by reducing the exposure of normal tissues to cytotoxic drugs. GDEPT seeks to increase the therapeutic index of anti- neoplastic agents by selective delivery or expression in cancer cells of a gene that encodes an enzyme that converts a relatively non- toxic prodrug into an anti- neoplastic agent.
Preferential activation of prodrugs at transduced cancer cells generates high drug concentrations in tumors while minimizing drug exposure to normal tissues. Several GDEPT systems have been developed for selective cancer treatment, the most prevalent being herpes simplex thymidine kinase activation of ganciclovir. Esherchia coli- derived cytosine deaminase activation of 5- fluorocytosine. Phosphorylation of ganciclovir by herpes simplex thymidine kinase is followed by a series of intracellular reactions that produces a triphosphate that is trapped within the target cells. The triphosphate product can compete with deoxyguanosine triphosphate during DNA elongation, thereby inhibiting DNA polymerase and causing single- strand breaks. Ganciclovir, however, is S- phase specific and kills only actively dividing cells,6 a disadvantage in tumors containing regions of non- proliferating cells.
Bystander killing by ganciclovir also relies on gap junctions for transport of the charged triphosphate to neighboring cells, which can limit the bystander effect in tumors since the expression of connexins is often decreased in neoplastic tissues and by hypoxia. Furthermore, low concentrations of ganciclovir can also cause chromosome breaks and sister chromatid exchange.
Cytosine deaminase derived from some bacterial and fungal sources is capable of converting the less toxic 5- fluorocytosine to 5- FU. FU) is enzymatically converted to 5- FUTP, which is incorporated into DNA and prevents nuclear processing of ribosomal and m.
RNA, and to 5- fluorouridine- 5'- monophosphate, which irreversibly inhibits thymidylate synthase. In contrast to ganciclovir, bystander killing by 5- FU is not dependent on gap junctions because this drug can diffuse into and out of cells. FC, however, is relatively hydrophilic and therefore diffuses across the plasma membrane of cells slowly, but is rapidly excreted from some cells. Furthermore, because the inherent sensitivity of tumor cells is an important factor in determining the efficacy of suicide gene therapy with 5- FC,1. Although suicide gene therapy with either ganciclovir or 5- FC appears to safe, only marginal clinical efficacy has been observed,1. GDEPT systems.- glucuronidase (G) is attractive for GDEPT for several reasons. First, a wide variety of glucuronide prodrugs are available, including prodrugs of effective anti- neoplastic agents such as doxorubicin,1.
In fact, glucuronide derivatives of almost any anti- neoplastic agent can be synthesized by employing linkers between the drug and glucuronide moieties. This is a major advantage since the most effective prodrug can be selected for a particular tumor type. Second, glucuronide prodrugs do not readily enter cells due to their charged carboxy group, minimizing interactions with endogenous G located inside lysosomes.
Thus, prodrug activation selectively occurs at sites of exogenous G expression only if the enzyme is located extracellularly. Surface expression of prodrug- activating enzymes may promote more extensive killing of non- transduced cancer cells since activated drug is not trapped in the cytosol of transduced cells. For example, hydrolysis of glucuronide prodrugs by G released from cells or targeted to the cell membrane produces potent cytotoxicity as well as bystander killing of neighboring enzyme- negative cells,2. Finally, glucuronide prodrugs produce potent anti- tumor activity in antibody- directed enzyme prodrug therapy (ADEPT), in which specific antibodies are employed to deliver G to cancer cells. G in the tumor interstitial space.
Although many variables may influence the effectiveness of GDEPT, we hypothesized that maximizing the activity of G on tumor cells is of primary importance. In the present study, we examined the effectiveness of anchoring G on cells with different juxtamembrane spacer domains and transmembrane domains (Figure 1a). As different sources of G display unique kinetic properties, we compared the surface expression, surface activity, in vitro prodrug activation and in vivo anti- tumor activity of E. G), murine (m. G), and human (h. G) G. h. G has previously been expressed as a secreted or membrane anchored form for the selective activation of a glucuronide prodrug of doxorubicin,2. G enzymes have not been examined.
Finally, we directly compared the in vitro and in vivo efficacy of two glucuronide prodrugs, HAMG and 9. ACG. HAMG is a glucuronide prodrug that can be enzymatically converted by G to p- hydroxy aniline mustard (p. HAM), a potent alkylating agent.
We previously demonstrated that combined treatment with immunoenzymes and HAMG could cure late- stage malignant ascites. ACG is a water soluble prodrug that can be hydrolyzed by G to release 9- aminocamptothecin (9. AC), an anti- tumor alkaloid that inhibits topoisomerase I to produce selective anti- tumor activity. ACG displays anti- tumor activity against human xenografts. The active prodrug of 9.
ACG is about 1. 00. HAMG, suggesting that these prodrugs may display differences in their effectiveness. Top of page. Materials and methods. Reagents and antibodies. The synthesis of p. HAM, HAMG, 9. AC and 9. ACG have been described.
Rat anti- HA antibody was from Roche (Basel, Switzerland). Mouse anti- -actin and rabbit anti- Bi. P were from Sigma Aldrich (St Louis, MO, USA). Horseradish peroxidase (HRP), FITC and rhodamine- conjugated secondary antibodies were from Jackson Immuno. Research Laboratories (Westgrove, PA, USA) or Organon Teknika Corp (Durham, NC, USA). Monoclonal antibody 1.
E8 (Ig. G1 against e. G) has been described. To generate monoclonal antibodies (m.
Ab) against h. G (1. B3, Ig. G1 and 7. G8, Ig. G2b), female Balb/c mice were s. G in complete Freund's adjuvant, and then boosted at 3–4 week intervals with decreasing doses of h. G in incomplete adjuvant.
At 3 days before fusion with FO myeloma cells, the mice were i. G in PBS. Hybridomas were screened by ELISA for clones secreting antibodies that bound h. G but not m. G or e. G. Stable hybridomas were obtained by repeated limiting dilution cloning.
A rat m. Ab against m. G (7. G7, Ig. G) was generated in an analogous fashion by immunizing Lou/c rats with recombinant m.
G before splenocytes were fused with FO myeloma cells. DNA constructs. The h.
G c. DNA fragment in p. LNCX- h. G,4. 0 the m. G c. DNA fragment in p. LHCX- m. G4. 0 and the e. G fragment in p. GUS N3. S (Clontech, Mountain View, CA, USA) were inserted in place of the 2. C1. 1 sc. Fv gene (Sfi.
I/Sal. I fragment) in p. C1. 1- PDGFR, p. 2C1. BGP- B7, p. 2C1. 1- e- B7, p. C1. 1- 1- B7 and p. C1. 1- CD4. 4- B7. G, m. G and e. G. Removal of the immunoglobulin hinge region in the 1 domain of p- h.
G- 1–B7 produced p- h. G- m. 1- B7. p- h. G- L- e- B7 includes c. DNA for a 1. 0 amino- acid linker (GGGGSGGGGS) at the 5'- end of the e- B7 domain.
G- ICAM was generated by inserting the h. G c. DNA fragment between the ICAM- 1 leader sequence and ICAM- 1 transmembrane domain in p. LTM- 1 (generously provided by Dr Alister Craig, University of Oxford, UK). Insertion of the h. G- e- B7, m. G- e- B7 and e.
G- e- B7 transgenes into the retroviral vector p. LNCX (BD Biosciences, San Diego, CA, USA) generated the retroviral vectors p. LNCX- h. G- e- B7, p. LNCX- m. G- e- B7 and p. LNCX- e. G- e- B7. Neo/GUS and p. LS/GUS, which direct the expression of e. G to the cytosol or allow secretion of e.
G, respectively, have been described. Expression of all transgenes was under control of the CMV promoter. Cell lines. Balb/3. T3 fibroblasts (CCL- 1.
CT2. 6 murine colon carcinoma cells (CRL- 2. DMEM (high glucose) supplemented with 1. HEPES, 2 g/l Na. HCO3, 1.
U/ml penicillin and 1. EJ human bladder carcinoma cells. RPMI containing the same supplements. The cells were free of mycoplasma as determined by PCR. Transfection of G transgenes. T3 fibroblasts were transfected with plasmid DNA using Lipofectamine 2.
Gibco Laboratories, Grand Island, NY, USA). To generate stable cell lines, p. LNCX- e. G- e- B7, p. LNCX- h. G- e- B7 and p.
LNCX- m. G- e- B7 were co- transfected with p. VSVG in GP2. 93 cells (Clontech) to produce recombinant retroviral particles. At 2 days after transfection, the culture medium was filtered, mixed with 8 g/ml polybrene and added to EJ or CT2. Stable cell lines were selected in medium containing G4. Western blot analysis.
Transiently transfected 3. T3 fibroblasts were boiled in reducing SDS buffer, electrophoresed on a SDS- PAGE and transferred to PVDF membranes. Membranes were sequentially probed with anti- HA antibody or m. Ab 1. B3 against h. G followed by HRP- conjugated secondary antibody. The membranes were stripped and reprobed with anti- -actin antibody.
Bands were visualized by ECL detection (Pierce, Rockford, IL, USA). Relative expression levels of G were normalized to - actin band intensities using the shareware program NIH Image (http: //rsb. Flow cytometer analysis. Cells were stained with anti- HA antibody followed by FITC- conjugated goat anti- rat F(ab')2 fragment. Alternatively, cells were stained with m. Ab 1. E8 against e. G,3. 9 m. Ab 7. G8 against h.
G or m. Ab 7. G7 against m.