Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro.

Gene electrotransfer is a safe and efficient nonviral technique for the transfer of nucleic acids of all sizes. Using a small reporter plasmid (3.5 kbp), electrotransfer of more than 90% of the cells, with ~70% viability, can be routinely achieved even in primary cells like mesenchymal stem cells. However, under the same experimental conditions, electrotransfer of larger plasmids (from 6 to 16 kbp) results in very low viability and transfection efficacy. Here, we show that these strong decreases are directly linked to the physical size of the plasmid molecule. Moreover, large plasmids are toxic only when the cells are exposed to electrotransfer pulses. This specific toxicity of large plasmids during electrotransfer is not due to transgene expression and occurs within less than 45 minutes. Indeed, postpulses recovery times of up to 45 minutes are able to entirely abolish the specific toxicity of large plasmid electrotransfer, resulting in a survival and transfection efficacy identical to that of small plasmids. Finally, electrotransfer of small and large plasmids can reach 90-99% of transfection with 60-90% survival considering the findings here reported.

Preactivated oxazaphosphorines designed for isophosphoramide mustard delivery as bulk form or nanoassemblies: synthesis and proof of concept.

Oxazaphosphorines are alkylating agents used in routine clinical practices for treatment of cancer for many years. They are antitumor prodrugs that require cytochrome P450 bioactivation leading to 4-hydroxy derivatives. In the case of ifosfamide (IFO), the bioactivation produces two toxic metabolites: acrolein, a urotoxic compound, concomitantly generated with the isophosphoramide mustard; and chloroacetaldehyde, a neurotoxic and nephrotoxic compound, arising from the oxidation of the side chains. To improve the therapeutic index of IFO, we have designed preactivated IFO derivatives with the covalent binding of several O- and S-alkyl moieties including polyisoprenoid groups at the C-4 position of the oxazaphosphorine ring to avoid cytochrome bioactivation favoring the release of the active entity and limiting the chloroacetaldehyde release. Thanks to the grafted terpene moieties, some of these new conjugates demonstrated spontaneous self-assembling properties into nanoassemblies when dispersed in water. The cytotoxic activities on a panel of human tumor cell lines of these novel oxazaphosphorines, in bulk form or as nanoassemblies, and the release of 4-hydroxy-IFO from these preactivated IFO analogues in plasma are reported.

Robust, efficient, and practical electrogene transfer method for human mesenchymal stem cells using square electric pulses.

Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with the ability to differentiate into various specific cell types, thus holding great promise for regenerative medicine. Early clinical trials have proven that MSC-based therapy is safe, with possible efficacy in various diseased states. Moreover, genetic modification of MSCs to improve their function can be safely achieved using electrogene transfer. We previously achieved transfection efficiencies of up to 32% with preserved viability in rat MSCs. In this study, we further improved the transfection efficiency and transgene expression in human MSCs (hMSCs), while preserving the cells viability and ability to differentiate into osteoblasts and adipocytes by increasing the plasmid concentration and altering the osmotic pressure of the electrotransfer buffer. Using a square-wave electric pulse generator, we achieved a transfection efficiency of more than 80%, with around 70% viability and a detectable transgene expression of up to 30 days. Moreover, we demonstrated that this transfection efficiency can be reproduced reliably on two different sources of hMSCs: the bone marrow and adipose tissue. We also showed that there was no significant donor variability in terms of their transfection efficiency and viability. The cell confluency before electrotransfer had no significant effect on the transfection efficiency and viability. Cryopreservation of transfected cells maintained their transgene expression and viability upon thawing. In summary, we are reporting a robust, safe, and efficient protocol of electrotransfer for hMSCs with several practical suggestions for an optimal use of genetically engineered hMSCs for clinical application.