Preliminary study on non-viral transfection of F9 (factor IX) gene by nucleofection in human adipose-derived mesenchymal stem cells

Abstract

Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment.Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX) where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen.Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver) where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration.Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia.

Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells

“…The transfection efficiency of our protocol was comparable with some previous studies with primary cells 16,28 and eminently higher than that of studies with different kinds of stem cells and other transient transfection methods (Table 2). 24,25,27,41–43 According to flow cytometry analysis, our protocol meets clinically relevant number of cell suitable for tissue engineering and cell transplantation.…”

“…25–27 Physical methods are reported with high transfection efficiency. Among the different complicated and expensive physical methods such as magnet-mediated transfection, biolistic particle delivery, or microinjection, 28–33 we decided for electroporation that is relatively easy and cheap. Here an electrical field is applied to permeabilize the cells for DNA transfer.…”

“…Here an electrical field is applied to permeabilize the cells for DNA transfer. 22,28 Our protocol should aim for number of cells high enough for clinical applications and sustainable enough to be applied for cell tracking over a long time but with the least possible impact on cell biology.…”

Mesenchymal stem cells as carrier of the therapeutic agent in the gene therapy of blood disorders

“…In another study, in non‐obese diabetic mice, the acute hepatic injury is induced by acetaminophen. Administration of AD‐MSC‐FIX caused an increased plasma level of FIX after 48 hr of injection and this increase remained fixed for 2 weeks (Olmedillas López, Garcia‐Arranz, Garcia‐Olmo, & Liras, ).…”

Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications

“…We identified appropriate NF conditions that, in our 5% SRGF expanded ASC, allowed higher transfection efficiency coupled with increased cell viability, in turn leading to a significantly elevated yield of modified ASC. A different program was previously [ 17 , 41 ] defined as suitable for the ASC transfection; such discrepancy can be ascribed to the different cell buffer used for electric shock administration, as well as to MSC origin [ 41 , 43 ] or to the cell donor age [ 17 ]. Here, we also showed that increasing SRGF availability in cell expansion medium over 5% failed to provide beneficial effects on cell transfection performance.…”