Vectofusin-1 Promotes RD114-TR-Pseudotyped Lentiviral Vector Transduction of Human HSPCs and T Lymphocytes

Abstract

Ex vivo transduction of human CD34+ hematopoietic stem/progenitor cells (hCD34+ HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34+ HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34+ HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34+ HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34+ HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering

“…An alternative method might be transduction based on vectofusin-1, a synthetic nontoxic histidine-rich peptide that promotes T-cell transduction with lentiviral vectors. 49 This soluble GMP-compliant enhancer could be easily added in advanced Prodigy settings for NK cell transduction. Time-lapse tracking experiments performed by fluorescent microscopy confirmed the results of expanded CAR NK cell cytotoxicity and demonstrated redirected E/T cell contacts of anti-CD123 CAR NK cells toward CD123-positive AML cells and subsequent apoptotic and necrotic reactions in the attacked AML cells.…”

Materials promoting viral gene delivery

“…Studies have shown enhanced TE of Vectofusin-1 compared to other delivery agents such as Tat, Pen, LAH4 derivatives, KH27K, R8, FUSO, polybrene, and retronectin. 328,[351][352][353][354][355] Interestingly, a variation in the histidine sequence order or peptide length resulted in a significant change of bioactivity. A minimum length of 21 amino acids of the LAH4 peptide family was found to be necessary for successful vector delivery.…”

High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia

“…Vectofusin-1, also known as LAH4-A4, is a cationic peptide derived from the LAH4 peptide family (47). Initially, LAH4 peptides were used as DNA transfection agents (48), but recent works showed the ability of Vectofusin-1 to promote transduction of human CD34 + hematopoietic stem and progenitor cells (hCD34 + HSPCs) and human T cells using a broad range of lentiviral and gammaretroviral pseudotypes, including RD114-TR and at least for hCD34 + HSPCs also VSV-G (47,49,50). Thereby, Vectofusin-1 was at least as effective as Retronectin in enhancing transduction, and in some cases more effective (47,50).…”

“…Initially, LAH4 peptides were used as DNA transfection agents (48), but recent works showed the ability of Vectofusin-1 to promote transduction of human CD34 + hematopoietic stem and progenitor cells (hCD34 + HSPCs) and human T cells using a broad range of lentiviral and gammaretroviral pseudotypes, including RD114-TR and at least for hCD34 + HSPCs also VSV-G (47,49,50). Thereby, Vectofusin-1 was at least as effective as Retronectin in enhancing transduction, and in some cases more effective (47,50). The two substances act differently enhancing gene delivery into target cells: Retronectin on the one hand is a fibronectin fragment that facilitates colocalization of viruses and target cells by binding viral particles and target cells with different domains (51).…”