Vesicular Stomatitis Virus G Research Articles

Intracellular Trafficking and Secretion of Adiponectin Is Dependent on GGA-coated Vesicles

Adiponectin (Acrp30) is an insulin-sensitizing hormone produced and secreted exclusively by adipose tissue. Confocal fluorescent microscopy demonstrated the colocalization of adiponectin with the Golgi membrane markers p115, beta-COP, and the trans-Golgi network marker, syntaxin 6. Treatment of cells with brefeldin A redistributed adiponectin to the endoplasmic reticulum where it colocalized with the chaperone protein BIP and inhibited secretion of adiponectin demonstrating a requirement for a functional Golgi apparatus for adiponectin release. Confocal fluorescent microscopy also demonstrated a colocalization of endogenous adiponectin with that of expressed GGA1myc (Golgi-localizing gamma-adaptin ear homology ARF-binding protein) but with no significant overlap between adiponectin and the GGA2myc or GGA3myc isoforms. Consistent with confocal fluorescent microscopy, transmission electron microscopy demonstrated the colocalization of GGA1 with adiponectin. Although GGA1 did not directly interact with the adiponectin protein, the adiponectin enriched membrane compartments of adipocyte were precipitated by a GST-GGA1 cargo binding domain (VHS) fusion protein but not with a GST-GGA2 VHS or GST-GGA3 VHS fusion proteins. Moreover, co-expression of adiponectin with a GGA1 dominant-interfering mutant (GGA1-VHS GAT domain) resulted in a marked inhibition of adiponectin secretion in both 3T3L1 adipocytes and HEK293 cells, whereas no inhibition was detected with the truncated mutants GGA2-VHSGAT or GGA3-VHSGAT. Moreover, co-expression of wild type GGA1 with adiponectin enhanced secretion of adiponectin. Interestingly, leptin secretion was unaffected by neither the wild type form or GGA1 mutant. Taken together these data demonstrate that the trafficking of adiponectin through its secretory pathway is dependent on GGA-coated vesicles.

354. Genetic Modification of Murine Stromal- Derived Cells (MSC) in Suspension Culture as a Model for the Rapid Generation of Transplantable Cytoreagents

Bone marrow-derived mesenchymal stromal cells (MSC) can contribute to tissue repair after cardiovascular injury, and appear to have immunomodulatory activity in some experimental models. Convenient recovery, propagation, and extensive proliferation characteristics recommend them for a range of cellular therapies, some of which require their stable genetic modification. Others have previously demonstrated that HIV-derived lentiviral vectors efficiently transduce human mesenchymal cells (Zhang XY et al., Mol Ther. 2002 May;5:555|[ndash]|65). However, adherence and ongoing subculture of MSC risk differentiation and may increase phenotypic heterogeneity within the cell product. Our studies explored whether stromal-derived cells can be efficiently transduced without adherence and subculture requirements. Murine (m-) MSC were established from C57BL/6 animals and characterized to ascertain differentiation capacity along adipogenic and osteogenic phenotypes. Cells were transduced with 3rd generation lentiviral vectors expressing GFP from an elongation factor 1 alpha promoter and pseudotyped with vesicular stomatitis virus G (VSV-G) protein. We trypsinized and washed mMSC from subconfluent primary cultures and transduced them in suspension or adherent after replating with subsequent determination of gene transfer, fluorescence intensity, and proviral copy numbers. Results demonstrated that gene transfer in suspension is sensitive to changes in vector- and fibronectin concentration, and routinely exceeded transfer to mMSC exposed in standard adherent culture. We were able to transduce up to 58% of cells after a single 1 hour incubation with vector at multiplicity of infection (MOI) of 10. Consistent with studies in murine fibroblasts, fluorescence intensity tended to increase with MOI. This was mirrored by a rapid rise in proviral copy number (up to 16 copies at MOI 10), determined by real-time PCR. We conducted experiments in mMSC (5th passage) immediately upon thawing, again showing gene transfer in over 50% of target cells. We further confirmed gene transfer rates of up to 35% in mMSC progenitors from day 14 initiation culture - commonly termed CFU fibroblast (CFU-F)- after a single vector exposure (MOI 10) of murine whole bone marrow immediately upon harvest. Gene transfer and CFU-F clonogenicity after transduction in suspension decreased at MOI greater than 3, suggesting VSV-G Env protein toxicity, previously described by others.

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.

Lentiviral vector–mediated, in vivo gene transfer to the tracheobronchial tree in fetal rabbits

Background/Purpose Fetal gene replacement is a novel, potential therapy for antenatally diagnosed monogenic disorders. The purpose of this study was to evaluate in vivo techniques of lentiviral (LV) vector–mediated gene transfer to the tracheobronchial tree in a rabbit model of fetal gene therapy. Methods Via triple plasmid transfection, vesicular stomatitis virus-G–pseudotyped LV vector containing green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter was constructed. In vivo gene transfer of 5 × 10 6 LV particles to fetuses of time-mated NZW rabbits (term = 31 days) was attempted using 2 techniques: (1) direct amniotic injection (gestation = 24-26 days) and (2) direct tracheal injection (gestation = 26 days). Injected fetuses and saline-injected littermate controls were delivered and killed on gestational day 30. Fetal and maternal tissues were analyzed. Results Both in vivo techniques produced gene transfer to fetal tissues (trachea, lung, liver, intestine), including those of some controls. In one prep, GFP DNA was identified in maternal lung. Conclusions Lentiviral vector–mediated GFP gene transfer to fetal rabbit tracheobronchial epithelium occurs within 4 days of transfection by both amniotic injection or direct fetal tracheal injection. This in vivo model confirms bioavailability of vector through amniotic fluid with some cross-infection of adjacent fetuses. Vector access to fetal tissues appears to be by both luminal and hematogenous routes. Transplacental gene transfer from fetus to mother may occur in this model.

Sustained elevation of neutrophils in rats induced by lentivirus‐mediated G‐CSF delivery

Patients with severe chronic and cyclic neutropenia, characterized by neutrophil numbers <500 cells/microl, are treated daily with recombinant granulocyte colony-stimulating factor (G-CSF). As an alternative delivery approach we investigated the ability of lentivirus vectors to provide sustained G-CSF expression. Fischer rats were injected intramuscularly (IM) with vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus pRRL-CMV-G-CSF-SIN that encoded rat G-CSF cDNA regulated by the human cytomegalovirus (CMV) promoter and incorporated a self-inactivating (SIN) construct in the 3' long terminal repeat (LTR). Control rats received normal saline or lentivirus encoding the enhanced green fluorescent protein (eGFP). Rats were serially monitored for blood cell production and tissues assayed for provirus distribution. Rats receiving a single IM injection of lentivirus exhibited elevated neutrophil counts for 14 months. Virus administration of 6 x 10(7) infectious units induced sustained levels of neutrophil production having a mean +/- standard deviation (SD) of 5650 +/- 900 cells/microl and rats that received a 10-fold lower dose of virus showed mean neutrophil counts of 3340 +/- 740 cells/microl. These were significantly higher than the mean of control animals receiving saline or control lentivirus (1,760 +/- 540 cells/microl, P < 0.0001). White blood cell (WBC) counts were significantly elevated in treated over control animals (P < 0.0001). Hematocrits (P > 0.3), lymphocytes (P > 0.2) and platelets (P > 0.1) were not significantly different between control and treated animals. Genomic DNA from muscle at the injection sites was positive for provirus, whereas lung, spleen, liver, kidney and non-injected muscle samples were all negative, suggesting lack of virus spread. These studies indicate that lentivirus vectors administered IM provide sustained, therapeutic levels of neutrophils and suggest this approach to treat patients with severe and cyclic neutropenia.

RNA interference-mediated silencing of the syntaxin 5 gene induces Golgi fragmentation but capable of transporting vesicles

It has been suggested that syntaxin 5 (Syx5) participates in vesicular transport. We examined the effects of Syx5 down-regulation on the morphology of the Golgi apparatus and the transport of vesicles in mammalian cells. Knockdown of the Syx5 gene resulted in Golgi fragmentation without changing the level of endoplasmic reticulum (ER)-resident proteins, other Golgi-SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors), and coatmer proteins. Strikingly, a major decrease in Syx5 expression barely affected the anterograde transport of vesicular stomatitis virus G (VSVG) protein to the plasma membrane. These results suggest that Syx5 is required for the maintenance of the Golgi structures but may not play a major role in the transport of vesicles carrying VSVG between the ER and the Golgi compartment.

493. Concentration and Purification of Lentivirus Pseudotypes Bearing Alternative Envelope Glycoproteins

Current problems that impact the use of lentiviral (LV) vectors for applications in large animals include low titers, poor purity and difficulties related to their large-scale production. A number of useful protocols are available to concentrate and purify LV vector pseudotypes bearing the traditional vesicular stomatitis virus G (VSV-G) glycoprotein. These protocols include centrifugation-based approaches, ultrafiltration, anion exchange chromatography, and size exclusion chromatography. There is an increasing demand for alternative (i.e. non-VSV-G-based) LV pseudotypes and thus there are needs for improved concentration/purification protocols for such pseudotypes. We have investigated scaleable protocols for the capture and purification of alternative LV pseudotypes based on anion exchange membrane chromatography. In one approach, HIV-1-based LV vectors pseudotyped with a lyssavirus (rabies-related) glycoprotein were generated by transient transfection. In another approach, amphotropic LV pseudotypes were generated using the STAR-A amphotropic packaging cell line. Virus-containing cell culture supernatants were subsequently captured onto chromatography membranes bearing functional quarternary (Q) ammonium groups and eluted with high salt and then desalted and further concentrated by ultrafiltration. Using this procedure, up to 87 % of the virus applied (up to 1.8 |[times]| 108 transducing units) could be recovered from a single anion exchange membrane unit with a membrane bed volume of 0.18 ml. Our results indicate that these purification procedures are useful for concentrating LV vectors bearing alternative envelope glycoproteins with a minimal loss of infectivity. We expect these approaches to facilitate the preparation of lentiviral vectors for large-scale preclinical studies. They may ultimately be applicable to generate clinical-grade LV vectors.

Discovery of Small-Molecule Human Immunodeficiency Virus Type 1 Entry Inhibitors That Target the gp120-Binding Domain of CD4

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.

Enhanced function with decreased internalization of carboxy-terminus truncated CXCR4 responsible for WHIM syndrome

WHIM (warts, hypogammaglobulinemia, recurrent bacterial infection, myelokathexis) syndrome is an autosomal dominant immune deficiency with severe chronic neutropenia and marrow neutrophil apoptosis. Carboxy-termini truncating mutations in the chemokine receptor CXCR4 have been identified in WHIM patients. We created a retrovirus encoding mutated CXCR4 (truncating point mutation 1000C-->T [R334X] inherited heterozygously in several WHIM patients) in order to transducer healthy human CD34 stem cells and K562 to overexpress mutated CXCR4 and determined its effect on receptor responses to stromal-derived factor-1 (SDF1). Retrovirus vector was engineered to coexpress WHIM-associated R334X mutated CXCR4 together with green fluorescent protein (GFP). Control vectors included similar constructs with wild-type CXCR4 (WT-CXCR4) or only GFP. CD34+ cells and K562 were transduced with these vectors. Populations of 100% transduced K562 were established by sorting GFP+ cells by flow cytometry. We performed migration and calcium flux assays of transduced CD34+ cells and transduced/sorted K562. We also examined receptor recycling in response to SDF1. Healthy human CD34+ cells and/or human erythroleukemia K562 cells transduced to express mutated CXCR4, WT-CXCR4, or GFP alone demonstrated that mutated CXCR4 was associated with enhanced calcium flux and enhanced migration. There was also decreased receptor internalization and enhanced recovery of surface mutated CXCR4 in response to SDF1 compared with WT-CXCR4. We propose that decreased internalization of WHIM-associated mutated CXCR4 leads to prolongation/enhancement of signaling in response to SDF1 and that this may provide the biochemical basis for the autosomal dominant abnormalities of cell trafficking and function associated with WHIM syndrome.

Comparison of HIV-1 and EIAV-based lentiviral vectors in corneal transduction

In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid system. We show that EIAV is more efficient than HIV in transducing human and rabbit corneal endothelial cells. Rabbit corneal endothelial cells are transduced in higher quantity than human corneal endothelial cells. In the inducible system, however, we detected impairment between the vector and its internal E-Selectin promoter. Instead of controlled transgene expression or silencing of promoter activity, the U3-modified long-terminal-repeats (LTR) impaired the conditional activity of the E-Selectin promoter. Significant transgene expression was seen without stimulation of the inducible promoter. We show efficient transduction by lentiviruses of a corneal endothelial cell line and of full thickness corneas from different species, confirming that those vectors would be appropriate tools for gene therapy of selected corneal diseases. However, the modification within the U3-LTR did not adequately allow regulated transgene expression. These findings have important implications for vector design for diagnostic or therapeutic opportunities.

Cholesterol and Fatty Acids Regulate Dynamic Caveolin Trafficking through the Golgi Complex and between the Cell Surface and Lipid Bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein

A novel and rapid method for the total particles quantification of murine leukemia virus derived retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein was developed using high performance liquid chromatography. Virus particles were detected by absorbance at 260 nm and quantified using a calibration curve generated from highly purified and concentrated viral stock characterized by negative stain electron microscopy. The method requires Benzonase ® digestion and concentration of the supernatant prior to analysis. The virus eluted in 12.55 min at a flow rate of 1 mL/min in 20 mM Tris–Cl, pH 7.4 + 1.1 M NaCl. The limits of detection and quantification of this assay were 4.71 × 10 8 and 1.57 × 10 9 viral particles/mL, respectively. Linearity was between 3.0 × 10 9 and 1.0 × 10 11 viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 × 10 −6. The assay precision was < 5% and <10% for intra- and inter-day analysis, respectively. This assay was used for the total particles quantification of a 7-day, large-scale perfusion culture production of a retroviral vector grown in 293 cells expressing the β-galactosidase gene.

ABC transporter inhibitors that are substrates enhance lentiviral vector transduction into primitive hematopoietic progenitor cells

AbstractHigh gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34+ cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors. (Blood. 2004;104:364-373)

730. Lentiviral Vectors Pseudotyped with Envelope Glycoproteins Derived from Human Parainfluenza Virus Type 3

Retroviral and lentiviral vectors are highly efficient in gene transfer. Their tropism, stability and ability to be concentrated by ultracentrifugation were greatly increased by pseudotyping with the vesicular stomatitis virus G (VSV-G) protein. This allowed expanded use of these vectors for many purposes. Although most current gene therapy studies focused on ex vivo gene transductions with these vectors, the next desirable step is to be able to inject them directly for systemic delivery. While vectors pseudotyped with VSV-G from Indiana serotype yielded high levels of gene transfer, successive rounds of their in vivo administration will have immunological limitations because this protein is known to elicit strong neutralizing antibodies. Hence, for the purpose of repeated administrations, additional non-crossreactive envelope proteins that have comparable value in pseudotyping retro- and lentiviral vectors are needed. Furthermore, as VSV-G is highly fusogenic, there is cytotoxicity associated with its use via concentrated vector stocks. Accordingly, a less cytotoxic envelope protein for pseudotyping is also highly desirable. Taking these criteria into consideration, we tested envelope proteins from three different vesiculoviruses, namely VSV-New Jersey, Chandipura and Piry. A third generation lentiviral vector encoding GFP reporter gene was pseudotyped with each of these envelope G proteins in a transient transfection packaging system. The respective pseudotyped lentiviral vectors were used to transduce different mammalian cells including BHK-21, HeLa, Cos, Prostate cancer cell lines, as well as primary human CD34+ hematopoietic cells and macrophages derived in vitro. The transfection efficiencies were compared to the vector pseudotyped with VSV-G Indiana. Our results showed that the three new G proteins used were effective in pseudotyping the lentiviral vectors as assayed by GFP marker expression from transduced cells. Differential transduction levels were found between different cell types indicating variation in cell tropism between different envelopes. These studies established that envelope proteins from related vesiculoviruses are useful for pseudotyping lentiviral vectors and provide alternative non-crossreactive envelope proteins for in vivo administration of gene transfer vectors.

The transmembrane domain of vesicular stomatitis virus glycoprotein suffices to anchor HIV-1 envelope gp120 expressed by a recombinant vaccinia virus.

The shedding of HIV-1 glycoprotein gp120 results from the proteolytic cleavage of its precursor gp160 into gp120 and an anchoring gp41, to which gp120 is non-covalently attached. This report is directed toward the anchorage of gp120 expressed by three recombinant vaccinia virus (rvv): vPE16 expresses wild-type gp160; vvE13 the gp120-vesicular stomatitis virus G transmembrane and cytoplasmic tail (VSVGTMCT) fusion protein; and vvE14 the gp120 with 52 amino acids (aa) from the vector. In order to convert gp120 into an integral membrane protein, a gp120- VSVGTMCT chimerical gene fragment has been constructed and expressed in mammalian cells. This gp120 fusion protein expressed by an rvv, vvE13, has been shown to elicit better immunogenicity and protection against a gp160-expressing tumor cell line than the full-length envelope (env) glycoprotein gp160 in mice. The results show that gp120-VSVGTMCT expressed by vvE13 is not shed because it is membrane associated. The hydrophobic fragment of vesicular stomatitis virus G (VSVG) furnishes an anchor of the chimeric protein just as it does in the native VSVG.

Human fetal trachea-scid mouse xenografts: efficacy of vesicular stomatitis virus-G pseudotyped lentiviral-mediated gene transfer

Background/Purpose Postnatal gene transfer in respiratory epithelium has been inefficient, particularly in submucosal gland cells, the target cells for cystic fibrosis transmembrane regulator (CFTR) gene transfer in cystic fibrosis. The authors hypothesized that fetal tracheobronchial epithelium may be more receptive to gene transfer in that precursor cells of the respiratory epithelium may be more accessible. Methods Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral vector was first tested in human fetal tracheal organ culture then validated in a human fetal tracheal xenograft model in severe combined immunodeficiency (SCID) mice. Between 7 × 10 7 and 1.6 × 10 8 transducing units of lentiviral vector encoding the transgene LacZ under the control of the human CMV promoter were administered onto the lumenal surface of the xenografts (n = 6). Biopsy specimens were taken from the xenografts at one month (n = 1), 2 months (n = 2), 3 months (n = 1), 6 months (n = 4), and 9 months (n = 1) after vector administration. Analysis of transgene expression was performed on X-gal stained sections. Results Transgene expression was observed in 20.2% to 99% of the surface epithelial cells (mean, 70.8 ± 32.3% SD) and in 28.4% to 99% of the submucosal gland cells (mean, 68.5 ± 26.2% SD) out to 9 months after vector administration in the tracheal xenografts, equivalent to 63 weeks postconceptual age. No staining was seen in the controls. Conclusions Excellent gene transfer in human fetal tracheal xenografts after VSV-G pseudotyped lentiviral vector administration, which may result from more accessible target precursor cells during development, suggests the feasibility of fetal gene therapy for the treatment of congenital airway disease such as cystic fibrosis.

Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis.

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)(2) domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (deltapsi(m)), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)2 domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (Δψm), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

Optimization of vesicular stomatitis virus-G pseudotyped feline immunodeficiency virus vector for minimized cytotoxicity with efficient gene transfer

FIV-based lentiviral vector has shown a unique opportunity as an efficient gene delivery vehicle, especially to nondividing human cells. Here, we genetically reconstructed the FIV-based vector by serially deleting residual virus genes of gag and vif, leading to minimized cytotoxicity together with efficient virus production and gene transfer. The modified FIV- based vector was generated by transiently transfecting 293T cells with three plasmids of the gene transfer vector with minimal gag region, the packaging plasmid without vif and the VSV-G-expressing plasmid. The vector was routinely generated as many as 1×10 7 transducing particles per ml and easily concentrated by simple centrifugation. The cytotoxic effect significantly decreased in sensitive cells to FIV infection even at high multiplicity of infection (MOI), such as 500. Moreover, the transduction efficiency was consistently retained after cell cycle was arrested in a variety of human cells. Taken together, our results suggest that the modified VSV-G pseudotyped FIV-based vector efficiently transduce dividing and nondividing human cells with minimal cytotoxicity.

Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64.

Unlike oncoretroviruses, lentiviral vectors can insert large genes and can target both dividing and nondividing cells; thus they hold unique promise as gene transfer agents. To enhance target range, the native lentiviral envelope glycoprotein is replaced (pseudotyped) with vesicular stomatitis virus G (VSVG), and the genes of interest are packaged in nonreplicating vectors by transient transfection with three plasmids. However, because of cytotoxic effects of VSVG expression in producer cells (293T cells) it has been difficult to generate a packaging cell line, required for even modest scale-up of vector production. Here we introduce a pseudotyped lentivirus vector using the baculovirus GP64 envelope glycoprotein. Compared with VSVG, GP64 vectors exhibited a similar broad tropism and similar native titers. GP64-pseudotyped vectors were usually highly concentrated without much loss of titer. Because, unlike VSVG, GP64 expression does not kill cells, we generated 293T-based cell lines constitutively expressing GP64. Our results demonstrate that the baculovirus GP64 protein is an attractive alternative to VSVG for viral vectors used in the large-scale production of high-titer virus required for clinical and commercial applications.