Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions

BACKGROUND: HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. METHODS: Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RESULTS: RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. CONCLUSIONS: Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.     

The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events

BACKGROUND: Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. METHODS: Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector - target cell contact and adsorption periods were studied. MOI between 0-32 was assessed on commonly used cell lines as well as a new cell line. RESULTS: We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0-32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained. CONCLUSION: Several variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted.     

RGB marking with lentiviral vectors for multicolor clonal cell tracking

Cells transduced with lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. We have recently extended this principle of clonal cell marking by introducing the method of RGB marking, which makes use of the simultaneous transduction of target cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. In accordance with the additive color model, individual RGB-marked cells display a large variety of unique and highly specific colors. Color codes remain stable after cell division and can thus be used for clonal tracking in vivo and in vitro. Our protocol for efficient RGB marking is based on established methods of lentiviral vector production (3-4 d) and titration (3 d). The final RGB-marking step requires concurrent transduction with the three RGB vectors at equalized multiplicities of infection (1-12 h). The initial efficiency of RGB marking can be assessed after 2-4 d by flow cytometry and/or fluorescence microscopy.     

Targeted gene modification in mouse ES cells using integrase‐defective lentiviral vectors

Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase‐defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector‐mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild‐type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild‐type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 ± 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene‐manipulation in therapeutic applications. genesis 47:217–223, 2009. © 2009 Wiley‐Liss, Inc.     

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.     

AKT2基因短发夹结构RNA慢病毒载体的构建及鉴定

目的：构建丝/苏氨酸蛋白激酶2（AKT2）基因RNA干扰（RNAi）慢病毒载体。方法：利用公用网站按照RNAi序列设计原则,设计RNAi靶点序列并合成靶序列的Oligo DNA,退火形成双链DNA,与经MluI和ClaI进行酶切后的PLVTHM载体连接产生shRNA慢病毒载体。应用shRNA慢病毒载体转染293T细胞及U87细胞,测定病毒滴度,流式细胞仪测定U87细胞的转染效率,PCR及Western blot鉴定AKT2基因在U87细胞中的下调作用。结果：成功构建了shRNA-AKT2慢病毒载体,经测序与设计合成的靶向链完全一致。荧光显微镜下观察293T细胞感染效率大于90%,病毒滴度为3.59×107TU/ml;流式细胞仪测定对AKT2细胞的转染效率为86.93%。PCR测定shRNA载体感染U87细胞后AKT2的干扰效率为68%。Western Blot结果显示该慢病毒载体对AKT2的表达有较为显著的敲减作用。结论：成功构建了人胶质瘤细胞株AKT2基因RNAi慢病毒载体,为后续的体内外功能学试验创造了条件。     

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.     

Susceptibility of cell populations to transduction by retroviral vectors

Retroviral transduction efficiency is related to the multiplicity of infection and the physiological state of the target cells. It is generally not known what proportion of a cell population is susceptible to transduction. We used coinfection with two retroviral vectors containing the marker genes for green fluorescent protein and the truncated human nerve growth factor receptor. In the CD34+ cell line TF-1 or human primary CD34+ hematopoietic progenitor cells, it was found that cells transduced with one vector had a better than random chance of transduction by the other vector. A probability model was developed to estimate target cell susceptibility; susceptibility was calculated as the product of the proportions of transgene-positive cells divided by the proportion of double-positive cells. By using this relationship, it was found that susceptibility was related to the target cell type and culture conditions but not the retroviral titer or the retroviral packaging envelope protein used in this study. Cotransduction with two vectors is a relatively simple procedure that provides a means to assess the maximum transduction level possible in a given cell population.     

Polycation-mediated enhancement of retroviral transduction efficiency depends on target cell types and pseudotyped Env proteins: implication for gene transfer into neural stem cells

Polycations such as polybrene (PB) are routinely used for most retroviral vector-mediated gene transfer studies because they can increase the infectivity of retroviruses. However, it was not systematically determined if addition of the polycation is an essential prerequisite for all retroviral transductions. To test this, we measured the effects of the polycation on transduction efficiency using various combinations of target cells and pseudotyped viral envelope (Env) proteins. Here, we show polycations do not always increase retroviral transduction efficiency and that their enhancing effect depends on both the type of target cells and Env proteins. The findings presented here also suggest that high transduction rates can be achieved in primary neural stem cells in vitro and in vivo by choosing an appropriate Env protein for pseudotyping without using polycations which are potentially toxic to primary cells and may change the intrinsic characteristics of cells.     

Retroviral vectors for vaccine development: induction of HIV-1-specific humoral and cellular immune responses in rhesus macaques using a novel MLV(HIV-1) pseudotype vector

Retroviral vectors have yet not been tested for their potential as vaccines despite their frequent utilization in gene therapy allowing for highly efficient gene transfer into a number of cell types and their suitability for large-scale production in biotechnology. To investigate MLV-based vectors suitability for inducing immune response against HIV-1-antigens, we generated a MLV(HIV-1) pseudotype vector enabling CD4-specific transduction of HIV-1 genes env, vpu, tat and rev originating from the pathogenic SHIV-89.6P. Functional expression of the lentiviral genes in packaging cells, human and rhesus CD4+ target cells was demonstrated by various assays. Following highly efficient ex vivo transduction, up to 3.4x10(7) autologous, transfer vector-positive rhesus peripheral blood mononuclear cells (rhPBMCs) were re-inoculated into a rhesus macaque. Five weeks after the initial inoculation HIV-1 Env-specific antibodies were detected using ELISA. ELIspot-assay revealed the induction of a HIV-1 Rev and Env-specific CTL-response 7.5 weeks after immunization. Thus, these novel MLV(HIV-1) vectors facilitate efficient transduction and subsequent expression of HIV-1-genes in CD4-positive host cells. Induction of both humoral and cellular HIV-1-specific immune responses in vivo confirmed their potential as an effective HIV-1 vaccine to be further studied in SHIV/rhesus macaque model of lentivirus infection.     

Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications

Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.     

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.     

Dependence of adenovirus infectivity on length of the fiber shaft domain

One of the objectives in adenovirus (Ad) vector development is to target gene delivery to specific cell types. Major attention has been given to modification of the Ad fiber knob, which is thought to determine virus tropism. However, among the human Ad serotypes with different tissue tropisms, not only the knob but also the length of the fiber shaft domain varies significantly. In this study we attempted to delineate the role of fiber length in coxsackievirus-adenovirus receptor (CAR)- and non-CAR-mediated infection. A series of Ad serotype 5 (Ad5) capsid-based vectors containing long or short fibers with knob domains derived from Ad5, Ad9, or Ad35 was constructed and tested in adsorption, internalization, and transduction studies. For Ad5 or Ad9 knob-possessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/alphav integrin-expressing cells. Ad5 capids containing short CAR-recognizing fibers were affected in cell adsorption and infection. In contrast, for the chimeric vectors possessing Ad35 knobs, which enter cells by a CAR/alphav integrin-independent pathway, fiber shaft length had no significant influence on binding or infectibility on tested cells. The weak attachment of short-shafted Ad5 or Ad9 knob-possessing vectors seems to be causally associated with a charge-dependent repulsion between Ad5 capsid and acidic cell surface proteins. The differences between short- and long-shafted vectors in attachment or infection were abrogated by preincubation of cells with polycations. This study demonstrates that the fiber-CAR interaction is not the sole determinant for tropism of Ad vectors containing chimeric fibers. CAR- and alphav integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.     

Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-016-9945-6) contains supplementary material, which is available to authorized users.     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

Assessment of optimal transduction of primary human skin keratinocytes by viral vectors

BACKGROUND: Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. METHODS: For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. RESULTS: We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. CONCLUSIONS: Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes.     

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

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 γ-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.