Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

Prodrug suicide gene therapy for cancer targeted intracellular by mesenchymal stem cell exosomes.

The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.

Neural stem cell-mediated enzyme/prodrug therapy for glioma: preclinical studies.

High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)-expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non-tumor-bearing and orthotopic glioma-bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.

Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Implication of functional activity for determining therapeutic efficacy of suicide genes in vitro.

Gene-Directed Enzyme Prodrug Therapy (GDEPT), commonly known as suicide gene therapy, provides a selective approach to eradicate tumor cells that is currently considered as an alternate approach to conventional therapy for cancers due to its high efficacy. Herein, we have demonstrated functional activity of the cytosine deaminase (CD) and the hybrid cytosine deaminase-uracil phosphoribosyltransferase (CD-UPRT) suicide genes in transfected cell lines. We have monitored retention profiles of various metabolites that were formed during enzymatic conversion of the prodrug 5-flurocytosine (5-FC) using reverse phase HPLC method. Therapeutic effect of suicide genes was established by cell viability and toxicity assay, whereas apoptotic induction was confirmed by DNA fragmentation ELISA. Our results demonstrated that 5-FC/CD-UPRT-mediated apoptotic cell death was more than 5-FC/CD, which could be further potentiated with anticancer compound curcumin. Such results corroborated 5-FC/CD-UPRT in combination with curcumin as a better chemosensitization method.

Clinical pharmacology of antifungal compounds.

Prompted by the worldwide surge in fungal infections, the past decade has witnessed a considerable expansion in antifungal drug research. New compounds have entered the clinical arena, and major progress has been made in defining paradigms of antifungal therapies. This article provides an up-to-date review on the clinical pharmacology, indications, and dosage recommendations of approved and currently investigational therapeutics for treatment of invasive fungal infections in adult and pediatric patients.

Ex vivo breast cancer cell purging by adenovirus-mediated cytosine deaminase gene transfer and short-term incubation with 5-fluorocytosine completely prevents tumor growth after transplantation.

Peripheral blood progenitor harvests of breast cancer patients are contaminated with tumor cells, suggesting a potential role for these cells in the relapse after high-dose chemotherapy. Whereas physical purging methods do not eliminate contaminating tumor cells completely, pharmacological purging, although highly efficient, is hampered by a strong nonspecific toxicity toward hematopoietic progenitor cells. Taking advantage of the high efficiency of adenovirus-mediated gene transfer to epithelial cells, we selectively loaded breast cancer cells in vitro with a cytotoxic drug by gene transfer of the prodrug-converting enzyme cytosine deaminase (AdCMV.CD) and 5-fluorocytosine (5-FC). Despite the low dose of vector administered, limited exposure to 5-FC, and transplantation only of viable tumor cells into SCID mice, all animals that received cells treated in vitro with AdCMV.CD plus 5-FC were completely free of tumor development. These data show that the selective loading of tumor cells with AdCMV.CD/5-FC might be useful for purging of autografts.