No abstract

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.

Research applications and cell therapies involving genetically modified cells require reliable, standardized, and costeffective 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. (Blood. 2011;117(16): e171-e181) IntroductionThe feasibility of using genetically engineered cells for therapy in humans has been demonstrated using various cell types, including tumor cells, 1-3 lymphocytes, dendritic cells, 4,5 fibroblasts, 6,7 hematopoietic stem cells (HSCs), 8,9 and mesenchymal stem cells (MSCs). 10,11 Actual or potential applications are as diverse as immune gene therapy for the treatment of cancer, 1-3 cancer therapy with T cells expressing chimeric T-cell receptors, 12,13 the treatment of hereditary diseases, 8,9,[14][15][16] and a plethora of applications in regenerative medicine. 17 With the emerging field of induced pluripotent stem cells, research in genetically engineered cell therapies has reached yet another level of pace and dimension. Clinical applications will require efficient, reliable, standardized methods for cell manipulation. For optimized reproducibility and wide practicality in a decentralized manner, such methods should compose a minimum number of handling steps and be cost-effective and amenable to automation in a closed system.The goal of this work is to provide a novel methodology for performing genetic modification and cell isolation in a single standardized procedure, which we call "magselectofection" ( Figure 1). It integrates nanomagnetic cell separation, which is an approved clinical application, 18,19 and nanomagnetically guided nucleic acid delivery known as magnetofection. [20][21][22] For magneti...