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F L Cosset

Publications and source records attributed to F L Cosset.

At least 55 records · Page 3Linked to original sources
PubMed1996

Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity.

Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.

Amino Acid Sequence↗
PubMed1996

Comparison of efficiency of infection of human gene therapy target cells via four different retroviral receptors.

The relative efficiency of transduction of gene therapy target cells was measured for retroviruses bearing the envelopes of amphotropic murine leukemia virus (MLV-A), xenotropic murine leukemia virus (MLV-X), gibbon ape leukemia virus (GALV), feline leukemia virus subgroup B (FeLV-B), and the feline endogenous virus RD114. These viruses use various cell-surface receptors. Activated peripheral blood lymphocytes (PBL) and primary melanoma cultures were infected relatively poorly by MLV-X pseudotypes. RD114 pseudotypes infected PBL relatively well, whereas bone marrow progenitor cells were efficiently infected by all viruses. Helper-free virus bearing the envelopes of MLV-A, RD114, or GALV was similarly tested. All infected melanoma or bone marrow progenitor cells efficiently, whereas MLV-A was relatively inefficient for infection of PBL. The general utility of RD114 pseudotyped virus for gene delivery coupled with its resistance to inactivation by human serum makes this envelope the most suitable choice for in vivo gene therapy.

Base Sequence↗
PubMed1996

Sensitization of cells and retroviruses to human serum by (alpha 1-3) galactosyltransferase.

Mammalian C-type retroviruses are inactivated by human serum, following triggering of the classical complement cascade. This may have inhibited transmission to humans of C-type oncoviruses from other mammals. Indeed, the retroviruses human immunodeficiency virus and human T-cell leukaemia virus are resistant to human complement. Antibody-independent activation of human C1q, the first component of the classical pathway, by retroviral envelope proteins has been described. However, retroviruses produced from human cells are resistant to inactivation by human complement and human serum is known to contain antibodies directed against carbohydrates on retroviral envelopes. Gal(alpha 1-3)Gal terminal carbohydrates are expressed by most mammals but are absent in humans, which lack a functional (alpha 1-3)galactosyltransferase gene. Here, we demonstrate that anti-Gal(alpha 1-3)Gal antibodies in human serum inactivate retroviruses produced from animal cells. Expression of porcine (alpha 1-3)galactosyltransferase in human cells renders the cells and the retroviruses they produce sensitive to human serum.

3T3 Cells↗
PubMed1996

Transposable elements behavior following viral genomic stress in Drosophila melanogaster inbred line.

To analyze the behavior of endogenous transposable elements under genomic stress, a Drosophila melanogaster inbred line was submitted to three kinds of viral perturbations. First, a retroviral plasmid containing the avian Rous Associated Virus type 2 (RAV-2) previously deleted for the viral envelope coding gene (env) was introduced by P element transformation into the Drosophila genome. An insertion of this avian retroviral sequence was detected by in situ hybridization in site 53C on polytene chromosome arm 2R. Second, Drosophila embryos were injected with RAV-2 particles produced by cell culture after transfection with the retroviral plasmid. Third, the Drosophila melanogaster inbred line was stably infected by the sigma native virus. It appears that neither the offspring of the flies in which the viral DNA was found integrated nor those from the infected sigma flies showed copia or mdg1 element mobilization. Injection of the avian RAV-2 particles led, however, to the observation of somatic transpositions of mdg1 element on the 2L chromosome, the copia element insertion pattern remaining stable. Thus, endogenous transposable elements show more instability in sublines injected with exogenous viral particles than in a transgenic subline containing a foreign viral insert, all transposable elements not being equally sensitive to such genomic stress.

Animals↗
PubMed1996

Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU.

We previously reported a strategy to redirect the retroviral host range by expressing single-chain antibodies (S. J. Russell, R. E. Hawkins, and G. Winter, Nucleic Acids Res. 21:1081-1085, 1993) or ligands (F.-L. Cosset, F. J Morling, Y. Takeuchi, R. A. Weiss, M. K. L. Collins, and S. J. Russell, J. Virol. 69:6314-6322, 1995) at the N terminus of Moloney murine leukemia virus (MoMLV) surface proteins (SU). Although such chimeric envelopes were able to bind the new receptors, the transduction efficiency of retargeted viruses was generally low. We hypothesized that conformational rearrangements of envelope glycoproteins were not optimally triggered following binding, and to overcome these postbinding blocks, we have generated here a set of chimeric MoMLV-derived envelopes targeted to the Ram-1 phosphate transporter in which we have varied the spacing between the Ram-1-binding domain and the MoMLV SU. All of the recombinant envelopes were correctly expressed on virions, and all bound efficiently to Ram-1. However, the interdomain spacing greatly affected the efficiency of gene transfer by retroviral vectors that had bound to Ram-1 via their chimeric envelopes. Optimal interdomain spacing allowed a 100-fold-increased viral transduction via Ram-1 compared to our previous results.

3T3 Cells↗
PubMed1996

Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single-chain antibody fragment-envelope fusion proteins.

As an approach to cell targeting by retroviruses, the lack of which constitutes one major limitation of retroviral vector technology, we engineered the Moloney murine leukemia virus ecotropic envelope glycoprotein. When inserted between amino acids 6 and 7 of the latter, a single-chain antibody fragment (ScFv) specific for human major histocompatibility complex class I molecules was shown to be able to redefine the tropism of ecotropic Moloney murine leukemia virus-derived retroviral particles by allowing infection of major histocompatibility complex class I-positive human cells. At variance with other recently described experimental systems, the type of modification adopted here allowed targeted infection in the absence of coexpressed wild-type env-encoded protein molecules. Interestingly, the chimeric ScFv-env protein also retained the ability to recognize the ecotropic receptor and allowed infection of murine cells, albeit at a reduced efficiency.

3T3 Cells↗
PubMed1996

TM domain swapping of murine leukemia virus and human T-cell leukemia virus envelopes confers different infectious abilities despite similar incorporation into virions.

We investigated the influence of transmembrane protein (TM) domains on incorporation of retroviral envelopes into virions and on infectivity. We introduced complete, truncated, or chimeric Friend murine leukemia virus (F-MuLV) and human T-cell leukemia virus type 1 (HTLV-1) envelopes into an MuLV particle-producing complementation cell line. As shown previously for HTLV-1 envelopes containing extracellular domains of F-MuLV TM (C. Denesvre, P. Sonigo, A. Corbin, H. Ellerbrok, and M. Sitbon, J. Virol. 69:4149-4157, 1995), reverse chimeric F-MuLV envelopes containing the extracellular domain of HTLV-1 TM were not processed. In contrast, a chimeric MuLV envelope containing the entire HTLV membrane-spanning and cytoplasmic domains (FHTMi) was efficiently processed, fusogenic as tested in a cell-to-cell assay, and efficiently incorporated into MuLV particles. However, these MuLV particles bearing FHTMi envelope proteins could not infect mouse or rat cells which are susceptible to wild-type F-MuLV. Therefore, envelopes which are readily fusogenic in cell-to-cell assays and also efficiently incorporated into virions may not necessarily confer virus-to-cell fusogenicity. HTLV envelopes, whether parental, chimeric (containing the MuLV cytoplasmic tail) or with a truncated cytoplasmic domain, were incorporated into MuLV particles with equal efficiencies, indicating that the cytoplasmic tails of these envelopes did not determine their incorporation into virions. In contrast to FHTMi envelope, HTLV-1 envelopes with F-MuLV membrane-spanning and cytoplasmic domains, as well as wild-type HTLV-1 envelopes, conferred virion infectivity. These results help to define requirements for envelope incorporation into retroviral particles and their cell-free infectivity.

3T3 Cells↗
PubMed1996

Homologous and nonhomologous retroviral recombinations are both involved in the transfer by infectious particles of defective avian leukosis virus-derived transcomplementing genomes.

We previously described avian leukosis virus-based packaging cell lines that produce stocks of retroviral vectors in which replication-competent viruses were not detectable. However, following infection of target cells with these retroviral stocks, we recently obtained colonies resulting from the transmission of recombinant genomes. Here, we have analyzed their genetic structure and shown that (i) each of them results from recombination between the packaging- and integration-defective transcomplementing genomes and the retroviral vector; (ii) recombination probably occurred during the reverse transcription step, involving strand switching of the reverse transcription growing point from the infectious retroviral vector to the transcomplementing RNA; and (iii) sequence identity and nonhomologous sequences were both used for the strand switching.

Animals↗
PubMed1996

Targeting of retroviral vectors through protease-substrate interactions.

Targetable, injectable vectors would greatly facilitate the development of in vivo therapy strategies. Viral and nonviral vectors can be targeted through ligand-receptor interactions, but protease-substrate interactions have not previously been exploited for vector targeting. Epidermal growth factor (EGF) was fused to a retroviral envelope glycoprotein via a cleavable linker comprising a factor Xa protease recognition signal. Vector particles displaying the cleavable EGF domain could bind to EGF receptors on human cells but did not transfer their genes until they were cleaved by factor Xa protease, whereupon gene delivery proceeded normally. Proteolytic activation of receptor-targeted vectors can therefore provide the basis for a novel two-step targeting strategy that may facilitate efficient targeted in vivo delivery of therapeutic genes.

3T3 Cells↗
PubMed1996

Targeting retrovirus entry.

Most of the clinical gene therapy trials that have been initiated to date have employed ex vivo strategies in which cells are genetically modified outside the body and reimplanted. The ability to deliver genes accurately and efficiently to selected target cell populations in vivo would greatly expand the scope of gene therapy, but current vectors are not well suited to this task. Here we review recent attempts to develop retroviral vectors incorporating engineered envelope glycoproteins that are capable of delivering their genes in a highly specific manner to selected human target cells.

Amino Acid Sequence↗
PubMed1995

Analysis of ALV-based packaging cell lines for production of contaminant defective viruses.

We have previously described avian leukosis virus-based packaging cell lines that express gag, pol, and env proteins from two transcomplementing genomes and produce helper-free stocks of retroviral vectors with different host ranges. In this report, we demonstrated that (i) despite the deletion of the psi packaging sequence, the packaging-defective transcomplementing retroviral transcripts were packaged into virions at a level that could reach 2.3% of a wild-type virus packaging level and (ii) despite deletion of the 3' LTR, these genomes were transferred along with the vector to target cells. As these genomes were also bearing a selectable gene, titers of the resulting contaminant particles could be estimated, depending on the cell line to be between 0 and 6 infectious particles/ml of supernatant.

Animals↗
PubMed1995

Defective retroviral endogenous RNA is efficiently transmitted by infectious particles produced on an avian retroviral vector packaging cell line.

This report describes the contamination of "helper-free" stocks of defective retroviral vector with particles bearing retroviral endogenous RNA. An avian leukosis virus-based packaging cell line was developed from LMH cells that bear the ev1, ev3, and ev6 retroviral endogenous loci. The results show that an endogenous retroviral transcript (ev3) was packaged into virions produced by this packaging cell line and was efficiently transferred along with the vector to target cells. The titer of the ev contaminant particles was estimated at 50-100 CA-p27gag-expressing units/ml of supernatant.

Animals↗
PubMed1995

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors.

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying the Neo(r) selectable marker and the Escherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%. Neo(r) and lacZ genes were transcribed in vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Animals↗
PubMed1995

Retroviral retargeting by envelopes expressing an N-terminal binding domain.

We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment.

Base Sequence↗
PubMed1995

High-titer packaging cells producing recombinant retroviruses resistant to human serum.

Novel retroviral protein expression constructs were designed to retain minimal retroviral sequences and to express dominant selectable markers by reinitiation of translation after expression of the viral genes. HT1080 cells were selected as producer cells for their ability to release high-titer viruses that are resistant to inactivation by human serum. Two HT1080-based packaging cell lines which produce Moloney murine leukemia virus cores with envelope glycoproteins of either amphotropic murine leukemia virus (FLYA13 line) or cat endogenous virus RD114 (FLYRD18 line) are described. Direct comparison with previous retroviral packaging systems indicated that 100-fold-higher titers of helper-free recombinant viruses were released by the FLYA13 and FLYRD18 lines.

Animals↗
PubMed1995

Structure and expression of endogenous retroviral sequences in the permanent LMH chicken cell line.

From DNA mapping data, four endogenous proviral loci have been observed in the chicken permanent cell line LMH. The locus corresponding to endogenous virus (ev) ev1 is present in duplicate whereas the locus corresponding to ev3 is present in one copy. The other loci are probably ev6 and a solitary long terminal repeat. A RNA Northern blot analysis revealed both ev3 and ev6 transcripts but no ev1 transcript was detected. Using avian leukosis virus (ALV)-based vectors, transcomplementing assays were performed. They demonstrate the correct expression and maturation of endogenous env proteins and the absence of production of functional gag and pol components, indicating that these cells are not competent for viral production.

Animals↗
PubMed1994

Type C retrovirus inactivation by human complement is determined by both the viral genome and the producer cell.

The inactivation of type C retroviruses by human serum may be a considerable impediment to the use of retroviral vectors in vivo for gene therapy. Here we show that virus inactivation is dependent both on the virus and on the cell line used to produce the virus. All viruses produced from murine NIH 3T3 or dog Cf2ThS+L- cells are sensitive to human serum. In contrast, those produced from mink Mv-1-Lu and human HOS or TE671 cells are at least partially resistant, with the exception of murine leukemia viruses. In particular, the feline endogenous virus RD114 is completely resistant to a panel of eight human sera when produced from Mv-1-Lu or HOS cells. This differential resistance is controlled by the viral envelope proteins. Virus inactivation can be correlated with the ability of the producer cells to be lysed by human serum. Inactivation of sensitive viruses requires the classical pathway of complement but does not require virion lysis.

3T3 Cells↗