Gene transfer into hCD34+ hematopoietic stem/progenitor cells (HSCs) using human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) has several promising therapeutic applications. Yet, efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist such as fibronectin fragments and cationic compounds, but all present limitations. In this study, we describe a new transduction enhancer called Vectofusin-1, which is a short cationic peptide, active on several LV pseudotypes. Vectofusin-1 is used as a soluble additive to safely increase the frequency of transduced HSCs and to augment the level of transduction to one or two copies of vector per cell in a vector dose-dependent manner. Vectofusin-1 acts at the entry step by promoting the adhesion and the fusion between viral and cellular membranes. Vectofusin-1 is therefore a promising additive that could significantly ameliorate hCD34+ cell-based gene therapy.

Gene delivery into hCD34؉ hematopoietic stem/progenitor cells (HSPCs) using human immunodeficiency virus, type 1-derived lentiviral vectors (LVs) has several promising therapeutic applications. Numerous clinical trials are currently underway. However, the efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist, such as fibronectin fragments or cationic compounds. Recently, we discovered Vectofusin-1, a new transduction enhancer, also called LAH4-A4, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 enhances the infectivity of lentiviral and ␥-retroviral vectors pseudotyped with various envelope glycoproteins. In this study, we compared a family of Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPC transduction enhancement with LVs pseudotyped with vesicular stomatitis virus glycoproteins and also with modified gibbon ape leukemia virus glycoproteins. By comparing the capacity of numerous Vectofusin-1 variants to promote the modified gibbon ape leukemia virus glycoprotein-pseudotyped lentiviral vector infectivity of HSPCs, the lysine residues on the N-terminal extremity of Vectofusin-1, a hydrophilic angle of 140°formed by the histidine residues in the Schiffer-Edmundson helical wheel representation, hydrophobic residues consisting of leucine were all found to be essential and helped to define a minimal active sequence. The data also show that the critical determinants necessary for lentiviral transduction enhancement are partially different from those necessary for efficient antibiotic or DNA transfection activity of LAH4 derivatives. In conclusion, these results help to decipher the action mechanism of Vectofusin-1 in the context of hCD34؉ cell-based gene therapy.

The transforming activity of SV40 large T-antigen (Tag) depends on its binding to cellular proteins involved in the control of the cell cycle (p53, pRb, p300..) and on the Jdomain region in the amino-terminus. We established transgenic lines expressing wild-type or Tag mutant proteins lacking one of the three transforming domains, to determine the respective contributions of these domains to hepatic tumour formation. Tag mutants with no pRb-binding domain or N-terminal fragment did not cause neoplastic liver abnormalities. The dl1137 Tag mutant protein, which inhibits pRb function without aecting p53, induced hepatic tumours. These tumours grew signi®cantly faster than those induced by wild-type Tag. Our results demonstrate dierent requirements for each of the inactivating functions of SV40 Tag in hepatocyte transformation and show that the loss of p53 function has only a moderate eect on hepatic tumour formation.

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.

The synthesis of a-tocopherol from 2,3-dimethylphytylquinol and Sadenosyl-L-methionine was achieved using Capsicum amwuan fruit chromoplasts. The enzymes involved in the cyclization (2,3-dimethyl-phytylquinol cyclase) and methylation (S-adenosyl methionine:y-tocopherol methyltransferase) are both localized in the chromoplast membrane fraction (envelopes and/or a-chlorophyll lamellae), in contrast to the stroma fraction.Terpenoid molecules play important structural and functional roles during plastid membrane organization (for a review, see Refs. 8 and 9). Among these molecules, the biosynthesis of atocopherol has been studied recently in isolated plastids (2,3,13,15). Based on the observed reactions, the following pathway has been proposed for the terminal steps (14, 17) 2,3-Dimethylphytylquinol -yT + SAMa T cyclization methylation However, in chloroplast subfractions, the cyclization reaction and the subsequent methylation of the endogenously preformed yT2 which yield aT have not been observed, although exogenously supplied yT is converted to aT by the plastid envelopes (13). Using Capsicum chromoplasts, we show that the plastid membranes (envelopes and a-Chl lamellae) are the sites of 2,3-dimethylphytylquinol cyclase and SAM:y-tocopherol methyltransferase activities which yield caT from dimethylphytylquinol. MATERIALS AND METHODSCapsicum annuum fruit chloroplasts were prepared according to a modified procedure (12, 18). Details will be given elsewhere. Capsicum chromoplasts were isolated from 2.5 kg of fruits according to a previously described method (3). The intact chromoplasts recovered at the 0.84 to 1.45 M interface of the gradient system were diluted (I ml suspension + I ml 50 mm Tris-HCI, pH 7.6, containing 10 ml DTT) and centrifuged at 4000g for 10 min. The pellet was homogenized in a loosely fitting Potter-Elvehjem homogenizer with 50 ml of a medium containing 10 mm DTT, 50 mm Tris-HCl (pH 7.6). The suspension was kept at 0 to 4°C for 10 min and centrifuged at 170,000g (R,,.) for 3.3 h in a precooled Beckman L3-50 equipped with a 42.1 rotor. At the completion, ' Supported by grants from the Mission de la Recherche and from the Centre National de la Recherche Scientifique (AI 03.1269).2 Abbreviations: yT, y-tocopherol; aT, a-tocopherol; SAM, S-adenosyl-L-methionine. the stroma was separated from the plastid membranes (chromoplast envelope and a-Chl lamellae) as revealed by the exclusive presence of galactosyltransferase activities in the chromoplast membranes and the phytoene synthetase activities in the stroma (4).The assay for a-tocopherol synthesis was based on the cyclization and methylation of 2,3-dimethylphytylquinol in the presence of S-adenosyl-L-[methyl-'4C]methionine (Amersham, France).

Human immunodeficiency virus type 1-derived lentiviral vectors (LVs) are becoming major tools for gene transfer approaches. Several gene therapy clinical studies involving LVs are currently ongoing. Industrial production of clinical-grade LVs is therefore an important challenge. Some improvements in LV production protocols have already been possible by acting on multiple steps of the production process like transfection, cell culture, or media optimizations. Yet, the effects of physicochemical parameters such as pH remain poorly studied. Mammalian cell cultures are generally performed at neutral pH, which may not be the optimal condition to produce high quantities of LVs with optimal infectious properties. In this study, we showed that lentiviral transient production in HEK293T cells is inversely dependent on the pH value of the harvesting medium. Infectious and physical titers of LVs pseudotyped with GALVTR or VSV-G glycoproteins are enhanced by two- to threefold at pH 6 compared with neutral conditions. pH 6-produced LVs are highly infectious on cell lines but also on relevant primary target cells like hCD34+ hematopoietic stem/progenitor cells. GALVTR-LV particles produced at pH 6 are highly stable at 37 °C and resistant to multiple freeze-thaw cycles. Higher levels of expression of intracellular pr55gag polyproteins are observed within HEK293T producer cells cultured at pH 6. The positive effect of pH 6 conditions is also observed for moloney-derived retroviral vectors produced from NIH-3T3 fibroblasts, arguing that the mildly acidic pH effect is not limited to the lentivirus genus and is reproducible in various producer cell lines. This observation may help us in the design of more effective LV production protocols for clinical applications.

With an increasing number of gene therapy clinical trials and drugs reaching the market, it becomes important to standardize the methods that evaluate the efficacy and safety of gene therapy. We herein report the generation of lentiviral standards which are stable, cloned human cells prepared from the diploid HCT116 cell line and which carry a known number of lentiviral vector copies in their genome. These clones can be used as reference cellular materials for the calibration or qualification of analytical methods that quantify vector copy numbers in cells (VCN) or lentiviral vector genomic integration sites (IS). Cellular standards were used to show the superior precision of digital droplet PCR (ddPCR) over quantitative PCR (qPCR) for VCN determination. This enabled us to develop a new sensitive and specific VCN ddPCR method specific for the integrated provirus and not recognizing the transfer plasmid. The cellular standards, were also useful to assess the sensitivity and limits of a ligation-mediated PCR (LM-PCR) method to measure IS showing that at least 1% abundance of a single IS can be detected in a polyclonal population but that not all IS can be amplified with similar efficiency. Thus, lentiviral standards should be systematically used in all assays that assess lentiviral gene therapy efficacy and safety.

Autophagy-related proteins such as Beclin-1 are involved in an array of complex processes, including antiviral responses, and may also modulate the efficiency of gene therapy viral vectors. The Tat-Beclin-1 (TB1) peptide has been reported as an autophagy-inducing factor inhibiting the replication of pathogens such as HIV, type 1 (HIV-1). However, autophagy-related proteins are also essential for the early steps of HIV-1 infection. Therefore, we examined the effects of the Beclin-1 evolutionarily conserved domain in TB1 on viral transduction and autophagy in single-round HIV infection or with nonreplicative HIV-1-derived lentiviral vectors. TB1 enhanced transduction with various pseudotypes but without inducing the autophagy process. TB1 augmented the transduction of human CD34+ hematopoietic stem/progenitor cells while maintaining their capacity to engraft into humanized mice. TB1 was as effective as other transduction additives and functioned by enhancing the adhesion and fusion of viral particles with target cells but not their aggregation. We also found that the N-terminal L1 loop was critical for TB1 transduction-enhancing activity. Interestingly, the Tat-Beclin-2 (TB2) peptide, derived from the human Beclin-2 protein, was even more potent than TB1 in promoting viral transduction and infection. Taken together, our findings suggest that the TB1 and TB2 peptides enhance the viral entry step. Tat-Beclin peptides therefore represent a new family of viral transduction enhancers for potential use in gene therapy.