Systemic TNFα gene therapy synergizes with liposomal doxorubicine in the treatment of metastatic cancer.

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Structure-activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) amides.

Sequence defined oligo (ethane amino) amides produced by solid-phase supported synthesis using different building blocks and molecular shapes were tested for structure-activity relationships in siRNA delivery. Efficient reporter gene knockdown was obtained in a variety of cell lines using either branched three-armed structures, or lipid-modified structures with i-shape, T-shape, U-shape configuration. For the majority of structures (apart from U-shapes), the presence of 2 or 3 cysteines was strictly required for polyplex stabilization and silencing activity. Although all four building blocks contain the ethylenediamine proton sponge motif, only oligomers assembled with the tetraethylenepentamine based amino acids (Stp, Gtp, Ptp) but not with the triethylenetetramine based amino acid (Gtt) were able to mediate efficient gene silencing. For the lipopolymeric structures, out of the tested saturated (from C4 to C18) and unsaturated (C18) fatty acid moieties, two proximate oleic acids or linolic acids provided the oligomers with the best gene silencing activity and also pH specific lytic activity at pH 5.5, presumably facilitating endosomal escape of the polyplexes. Evaluation of oligomer chain length revealed a minimal number of at least two oligo (ethane amino) building blocks per oligomer arm as necessary for the vast majority of structures, but only marginal changes were found with higher numbers (structures with up to 60 ethane amino nitrogens were evaluated). Two promising carriers (T-shape 49, i-shape 229) were also evaluated for EG5 siRNA delivery. This resulted in tumor cell cycle arrest, and appearance of mitotic monoastral spindles both in vitro and in vivo upon systemic delivery. Repeated intratumoral treatment with EG5 siRNA polyplexes significantly reduced Neuro2A-eGFPLuc tumor growth in a siRNA-specific manner.

Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells.

Research applications and cell therapies involving genetically modified cells require reliable, standardized, and cost-effective methods for cell manipulation. We report a novel nanomagnetic method for integrated cell separation and gene delivery. Gene vectors associated with magnetic nanoparticles are used to transfect/transduce target cells while being passaged and separated through a high gradient magnetic field cell separation column. The integrated method yields excellent target cell purity and recovery. Nonviral and lentiviral magselectofection is efficient and highly specific for the target cell population as demonstrated with a K562/Jurkat T-cell mixture. Both mouse and human enriched hematopoietic stem cell pools were effectively transduced by lentiviral magselectofection, which did not affect the hematopoietic progenitor cell number determined by in vitro colony assays. Highly effective reconstitution of T and B lymphocytes was achieved by magselectofected murine wild-type lineage-negative Sca-1(+) cells transplanted into Il2rg(-/-) mice, stably expressing GFP in erythroid, myeloid, T-, and B-cell lineages. Furthermore, nonviral, lentiviral, and adenoviral magselectofection yielded high transfection/transduction efficiency in human umbilical cord mesenchymal stem cells and was fully compatible with their differentiation potential. Upscaling to a clinically approved automated cell separation device was feasible. Hence, once optimized, validated, and approved, the method may greatly facilitate the generation of genetically engineered cells for cell therapies.