A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells

Abstract

Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic.

Chloroquine modulates inflammatory autoimmune responses through Nurr1 in autoimmune diseases

“…Finally, the viral titer was determined using the QuickTiter™ Lentivirus Titer Kit (Cell Biolabs) according to the manufacturer’s instruction. Lentivirus transduction into primary T cell was performed with Lentiboost (Sirion Biotech) 47 at multiplicity of infection (MOI) of 50 or 100, based on toxicity optimization. After 24 h in culture, cells were washed and then stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies.…”

Materials promoting viral gene delivery

“…In combination with polybrene the TE was further elevated, which was explained by the distinct modes of each adjuvant, polybrene compensating electrostatic repulsion and poloxamer 338 fluidization the membrane. 240 More recently, poloxamer 338 was commercialized as LentiBOOST™, and applied for LV delivery to CD34+ stem cells, [241][242][243] CD4+ and CD8+ stem cells, 244 and T-cells. 245 Enhanced TE, non-toxicity and clinical relevance were emphasized in all of these studies.…”

Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells

“…There are conflicting reports about the potential influence of lentiviral transduction on cell viability. Both negative influences [ 48 , 56 ] and no influences [ 57 ] of polybrene on cell proliferation have been reported after lentiviral transduction of primary cultures. Polybrene can disrupt the transmembrane potential in some sensitive cells [ 56 ].…”

“…Both negative influences [ 48 , 56 ] and no influences [ 57 ] of polybrene on cell proliferation have been reported after lentiviral transduction of primary cultures. Polybrene can disrupt the transmembrane potential in some sensitive cells [ 56 ]. We cannot rule out that the lower cell density observed in some of the cultures here can be ascribed to the use of polybrene in the transduction step…”