Concurrent Measures of Fusion and Transduction Efficiency of Primary CD34+ Cells with Human Immunodeficiency Virus 1-Based Lentiviral Vectors Reveal Different Effects of Transduction Enhancers

Abstract

Lentiviral vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking for precisely evaluating parameters that control the efficiency of transduction in relation to the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of nonreplicative recombinant LVs in various cell types, including primary human hematopoietic stem progenitor cells (HSPCs), and to quantify the level of transduction of the same initially infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low-affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm, which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing, for instance, the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers such as Vectofusin-1 or Retronectin can partially relieve the postentry block, but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols.

Simultaneous Analysis of Secondary Structure and Light Scattering from Circular Dichroism Titrations: Application to Vectofusin-1

“…These results are in good agreement with biological studies indicating that vectofusin-1 strongly promotes virus-cell adhesion1524. The lipid envelope of HIV-1 derived lentiviral vectors as well as eukaryotic cells (e.g.…”

“…This result is in excellent agreement with the increased virus-cell adhesion that is believed to be one of the major factors making vectofusin-1 such an efficient transduction enhancer. If the mechanisms described here and in the papers by D. Fenard15 and D. Ingrao24 are indeed the main factors underlying the vectofusin-1 efficacy as transduction enhancer, the method described here can be used in medium-throughput screening of peptide libraries for promising transduction enhancers by monitoring liposome self-association, liposome disruption and peptide structure and binding using a single CD titration.…”

Molecular Determinants of Vectofusin-1 and Its Derivatives for the Enhancement of Lentivirally Mediated Gene Transfer into Hematopoietic Stem/Progenitor Cells

“…After thawing, the survival rate of hCD34ϩ cells was evaluated using the trypan blue exclusion method. Next, preactivation of hCD34ϩ cells was performed overnight as described previously (2). Preactivated cells were plated in 96-well plates, and transduction was initiated by adding the desired amount of LV particles (2 ϫ 10 7 infectious genome/ml for highly purified VSV-G-LVs and 1-2 ϫ 10 6 transducing units/ml for GALVTR-LVs) mixed with or without LAH4 peptide derivatives (final concentration of 12 g/ml).…”

“…For LV applications relying on ex vivo transduction of hCD34ϩ hematopoietic stem/progenitor cells (HSPCs), viral entry represents a critical step: the adhesion and the fusion of viral particles with the plasma membrane of HSPCs (2). Viral entry efficiency is partially dependent on the envelope glycoprotein (GP) used to pseudotype LVs and, therefore, the relative paucity of viral receptors interacting with the GP of choice.…”

Syncytins enable novel possibilities to transduce human or mouse primary B cells and to achieve well-toleratedin vivogene transfer

“…VF1 is an efficient retroviral and lentiviral transduction enhancer with pseudotypes such as GALV, GALV-TR, RD114-TR or MLV-A as well as with VSVg which all use receptor-mediated endosomal entry. VF1 enhances LV transduction of target cells by enhancing the adhesion and fusion steps (46) and by promoting viral pull-down following formation of nanofibrils in medium (47). It is therefore interesting that VF1 also promotes the transduction of target cells mediated by syncytins considering the high fusogenic activity of these proteins, which we observed during production and which is not observed to this level with other glycoprotein LV pseudotypes.…”