Simultaneous infection with retroviruses pseudotyped with different envelope proteins bypasses viral receptor interference associated with colocalization of gp70 and target cells on fibronectin CH-296

Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.     

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector

BACKGROUND: Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. METHODS: Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. RESULTS: In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. CONCLUSIONS: In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use.     

Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein

Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34(+) (hCD34(+)) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34(+) cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34(+) cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.     

Infection of human hematopoietic progenitor cells using a retroviral vector with a xenotropic pseudotype

In an effort to determine if viral envelope type influences the infectivity of human hematopoietic progenitor cells with retroviral vectors, we have pseudotyped the retroviral vector N2, which confers G418-resistance, in either an amphotropic or xenotropic envelope. Vector titres obtained by the pseudotype procedure were nearly two orders of magnitude lower than the titer obtained when N2 was packaged using the amphotropic PA317 packaging cell line. Despite its low titer, xenotropically pseudotyped N2 generated G418-resistant hematopoietic colonies at levels approaching those observed after bone marrow was infected using vector packaged using PA317 cells. These results suggest that manipulations of vector envelope may lead to improvements in the level of infection of human hematopoietic stem cells.     

Entry of Amphotropic and 10A1 Pseudotyped Murine Retroviruses Is Restricted in Hematopoietic Stem Cell Lines

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70^SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70^SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.     

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer.

Recombinant retrovirus vectors are widely used for gene transfer studies. The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo. A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector. Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli beta-galactosidase-producing pseudotype and amphotropic vector. High rates of beta-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer. Subsequent studies demonstrated that beta-galactosidase protein was concentrated and copurified during pseudotype vector preparation, resulting in high rates of protein transfer rather than stable gene transfer, a process referred to as pseudotransduction. The cotransfer of protein with concentrated pseudotyped retroviruses indicates that caution must be used when interpreting gene transduction efficiencies in gene therapy experiments.     

Transduction of CD34+ hematopoietic progenitor cells with an antitat gene protects T-cell and macrophage progeny from AIDS virus infection.

Transduction of hematopoietic stem cells with genes that inhibit human immunodeficiency virus (HIV) replication has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an autoregulated gene, antitat, to inhibit replication of simian immunodeficiency virus (SIV) and HIV type 1 (HIV-1) in hematopoietic cells derived from transduced progenitor cells. The antitat gene expresses an antiviral RNA encoding polymeric Tat activation response elements in combination with an antisense tat moiety under the control of the HIV-1 long terminal repeat. CD34+ hematopoietic progenitor cells were transduced with a retroviral vector containing the antitat gene and then cultured under conditions that support in vitro differentiation of T cells or macrophage-like cells. Rhesus macaque CD4+ T cells and macrophage-like cells derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV, reflecting a 2- to 3-log reduction in peak SIV replication compared with controls. Similarly, human CD4+ T cells derived from CD34+ cord blood cells transduced with antitat were also resistant to infection with HIV-1. No evidence for toxicity of the antitat gene was observed in any of five different lineages derived from transduced hematopoietic cells. These results demonstrate that a candidate therapeutic gene introduced into hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication following T-cell differentiation and support the potential use of the antitat gene for stem cell gene therapy.     

Retroviral vector targeting to human immunodeficiency virus type 1-infected cells by receptor pseudotyping

We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell-tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression.     

Retroviral gene transfer inXenopus cell lines and embryos

Summary A new class of retroviral vector pseudotypes have an expanded host species range and can be concentrated to high titers by ultracentrifugation. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein. We tested (a) the ability of pseudotyped (pantropic) and unmodified (amphotropic) vectors to stably infect three diffeentXenopus laevis cell lines, including one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate foreign gene expression in the embryonic CNS. Expression of the neomycin phosphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amphotropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stage 10, 20, and 25 embryos microinjected in the blastocoel or neural tube cavities with concentrated pantropic vector (10⁸ cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector containing the coding sequence for the β-galactosidase gene into the neural tube lumen of 24-h embryos yielded β-galactosidase expressing cells in the brain. Thus, retroviral vectors provide an additional approach to existing strategies for gene transfer inXenopus embryos and cell lines.     

Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins

Bone-marrow-derived mesenchymal stem cells (MSCs) have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. We have designed and tested a series of human immunodeficiency virus type 1 (HIV-1)-based vectors and vectors based on the oncogenic murine stem cell virus to deliver and express transgenes in human MSCs. These vectors were pseudotyped with either the vesicular stomatitis virus G (VSV-G) glycoprotein (GP) or the feline endogenous virus RD114 envelope GP. Transduction efficiencies and transgene expression levels in MSCs were analyzed by quantitative flow cytometry and quantitative real-time PCR. While transduction efficiencies with virus particles pseudotyped with the VSV-G GP were found to be high, RD114 pseudotypes revealed transduction efficiencies that were 1 to 2 orders of magnitude below those observed with VSV-G pseudotypes. However, chimeric RD114 GPs, with the transmembrane and extracellular domains fused to the cytoplasmic domain derived from the amphotropic Moloney murine leukemia virus 4070A GP, revealed about 15-fold higher titers relative to the unmodified RD114 GP. The transduction efficiencies in human MSCs of HIV-1-based vectors pseudotyped with the chimeric RD114 GP were similar to those obtained with HIV-1 vectors pseudotyped with the VSV-G GP. Our results also indicate that RD114 pseudotypes were less toxic than VSV-G pseudotypes in human MSC progenitor assays. Taken together, these results suggest that lentivirus pseudotypes bearing alternative Env GPs provide efficient tools for ex vivo modification of human MSCs.     

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.     

Inhibition of HIV-1 replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells

The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA(i)(Met) promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4(+) cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.     

Gene delivery to airway epithelial cells in vivo: a direct comparison of apical and basolateral transduction strategies using pseudotyped lentivirus vectors

Lentivirus vectors are being investigated as gene delivery vehicles for cystic fibrosis airway gene therapy. Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped vectors transduce airway epithelia via receptors that are located predominantly on the basolateral surface of the airway epithelium. Effective transduction with VSV-G-pseudotyped vectors requires the use of a pre-treatment that disrupts epithelial tight junctions, allowing access to these basolateral receptors. In contrast, it has been reported that apically targeted lentiviral vectors allow efficient gene transfer in the absence of any pre-treatment. In a direct comparison of transduction by a VSV-G-pseudotyped vector, in combination with a pre-treatment with lysophosphatidylcholine (LPC), and the same vector pseudotyped with the apically targeted baculovirus GP64 envelope (without any pre-treatment), the GP64 vector was found to be significantly less efficient. However, when a pre-treatment with LPC was used the level of transduction with the GP64-pseudotyped lentiviral vector was not significantly different to that resulting from the VSV-G-pseudotyped vector. The cell types transduced with each vector were essentially the same, with the majority of cells transduced being respiratory (ciliated cells). However, unlike the VSV-G-pseudotyped vector, which results in persisting gene expression, transduction with the GP64-pseudotyped vector resulted in gene expression that declined to undetectable levels over six months, whether or not an LPC pre-treatment was used.     

逆转录病毒载体介导MGMT和MDR1基因增强人脐血CD34＋细胞对联合化疗抗性的研究

To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) and multidrug resistance gene (MDR1) increase resistance to 1,3-Bis(2-Chloroethy1)-1-Nitrosourea (BCNU) and P-glycoprotein effluxed drugs, the present authors obtained a full length cDNA fragment encoding MGMT from liver tissue of a patient with cholelithiasis by RT-PCR. A bicistronic retroviral vector G1Na-MGMT-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by electric performation method, using the medium containing VCR and BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD34+ cells were enriched with a high-gradient magnetic cell sorting system (MACS), and then transfected repeatedly with supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hemapoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, Western blot, FACS and MTT assay were used to evaluate the transfer and expression of the double genes in cord blood CD34+ cells. The cDNA encoding MGMT was verified by DNA sequencing and the bicistronic retroviral vector was confirmed by restriction endonuclease analysis. The purity of cord blood CD34+ cells was approximately 92% and recover rate was 75%, the highest titer of recombinant amphotropic retrovirus in the supernatant was up to 5.8 x 10(5) cfu/ml. The efficiency of gene transduction was 18% and 20% tested by colony formation and PCR, respectively. No helper virus was found by both nested PCR and rescue assay. The results showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The MTT analysis showed a 4.5 to 7.8-fold increase of resistance of transducted cells to BCNU and P-glycoprotein effluxed drug as compared with the nontransduced cells. This study provided a foundation for ameliorating combination chemotherapy toxicity in tumor clinical trial.     

Cholesterol supplementation during production increases the infectivity of retroviral and lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G)

Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl-beta-cyclodextrin (MbCD)) on the production of Molony murine leukemia virus (MoMuLV) retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than 2-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors.     

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.     

Vesicular Stomatitis Virus G-Pseudotyped Lentivirus Vectors Mediate Efficient Apical Transduction of Polarized Quiescent Primary Alveolar Epithelial Cells

We investigated the use of lentivirus vectors for gene transfer to quiescent alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) grown on plastic or as polarized monolayers on tissue culture-treated polycarbonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by confocal microscopy and quantified by fluorescence-activated cell sorting, was dependent on the dose of vector, ranging from 4% at a multiplicity of infection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a comparable titer and MOI, transduction of these cells by a similarly pseudotyped murine leukemia virus vector was approximately 30-fold less than by the lentivirus vector. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent AEC monolayers (R(t) > 2 kOmega. cm(2); MOI = 10) revealed efficient transduction only when VSV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment with EGTA to increase access to the basolateral surface did not increase transduction of apically applied virus, indicating that transduction was primarily via the apical membrane domain. In contrast, differentiated tracheal epithelial cells were transduced by apically applied lentivirus only in the presence of EGTA and at a much lower overall efficiency (approximately 15-fold) than was observed for AEC. Efficient transduction of AEC from the apical cell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that differences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.     

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.