EP1025208A2 - Verpackungszellinie zur herstellung von pseudotypisiertem mlv - Google Patents

Verpackungszellinie zur herstellung von pseudotypisiertem mlv

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EP1025208A2
EP1025208A2 EP98956852A EP98956852A EP1025208A2 EP 1025208 A2 EP1025208 A2 EP 1025208A2 EP 98956852 A EP98956852 A EP 98956852A EP 98956852 A EP98956852 A EP 98956852A EP 1025208 A2 EP1025208 A2 EP 1025208A2
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cell line
mlv
retroviral
packaging cell
siv
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French (fr)
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Stefano Indraccolo
Fabrizio Mammano
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Universita degli Studi di Padova
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Universita degli Studi di Padova
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13051—Methods of production or purification of viral material
    • C12N2740/13052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15051—Methods of production or purification of viral material
    • C12N2740/15052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae

Definitions

  • the present invention relates to retroviral particles carrying the envelopes of heterologous viruses that are discriminating in their binding to host cell membrane bound receptors.
  • ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use.
  • the ability to target the delivery of genes to predefined cell types is presently difficult, regardless of the method used for gene transfer.
  • the infection spectrum of enveloped viruses is determined by the interaction between viral surface (SU) proteins encoded by the retroviral gene, env, and host cell membrane proteins which act as receptors.
  • Vectors derived from viruses will deliver genes to the same cell types as the original virus does, unless the infection spectrum of the vector virus is modified.
  • retroviruses are derived from murine leukaemia virus (MLV); a retrovirus that is able to infect many different cell types. This is due to the expression of the cognate receptor or recognition site, i.e. the cationic amino acid transporter for rodent cells (ecotropic virus) (Kim, J.W., Closs, E.I., Albritton, L.M. and Cunningham, J.M. 1991 , Nature 352: 725-728; Wang, H., Kavanaugh, M.P., North,
  • MMV murine leukaemia virus
  • MLV has been the retrovirus of choice for the production of RVs, because of the capability of this virus to produce high titre systems, together with the fact that the MLV is a fairly simple virus, and that its biology is well understood.
  • Other retroviruses or enveloped viruses are less promiscuous than MLV in their infection spectrum, but also often give rise to lower titre systems. It is also, at least presently, difficult to construct vectors based upon these virus systems, in part due to the complex nature of their life cycles (G ⁇ nzburg, W. H. and Salmons, B. 1992, Biochem. J. 283, 625-632).
  • pseudotyped recombinant retroviruses consisting of the core and genome derived from well established MLV based retroviral vectors and the envelope of a second retrovirus or other enveloped virus that shows a limited infection spectrum.
  • pseudotyped viruses would have an altered infection spectrum, since they are able to infect the same cells as the second virus providing the envelope and/or surface proteins.
  • RVs have already been produced in the laboratory by a number of groups using packaging cell lines that produce gag and pol proteins from one virus and env proteins from a second virus.
  • VSV highly promiscous vesicular stomatis virus
  • GaLV SEATO-MoMuLV hybrid particles were generated at titers approximately equivalent to those obtained with the MoMuLV particles, and the infection spectrum correlates exactly with the previously reported in vitro host range of wild type GaLV SEATO, i.e. bat , mink, bovine and human cells.
  • the apparent titers of HTLV-I MoMuLV (1-10 CFU/ml) were substantially lower than the titers achieved with either the MoMuLV or GaLV-MoMuLV recombinant virions.
  • the HTLV-I hybrid virions were able to infect human and mink cells (Wilson, C, Reitz, M.S., Okayama, H., Eiden, M.V. 19898, Journal of Virology, Vol 63, No. 5, 2374- 2378).
  • pseudotyped retroviral particles carrying therapeutic genes will be useful in the treatment of various viral or bacterial infections, as well as cancer.
  • the invention then, inter alia, comprises the following , alone or in combination:
  • a packaging cell line packaging a MLV-based recombinant retroviral genome into a particle comprising a SIV envelope
  • the packaging cell line as above wherein said heterologous RNA and DNA, respectively, is selected from one or more elements of the group consisting of regulatory elements and/or promoters, regulatory elements and/or promoters that are target cell specific in their expression and/or regulatory elements that are regulatable by transacting molecules;
  • retroviral genome comprises at least one coding sequence selected from one or more elements of the group comprising marker genes, therapeutic genes, antiviral genes, antitumour genes, cytokine genes;
  • the packaging cell line as above wherein said coding sequence comprises additionally at least one non coding sequence selected from regulatory elements and/or promoters, regulatory elements and/or promoters that are target cell specific in their expression and/or regulatory elements that are regulatable by transacting molecules, said non coding sequences regulating the expression of at least one of the coding sequences of said retroviral genome; use of the packaging cell line as above for production of pseudotyped retroviral particles;
  • the pseudotyped retroviral as above comprising a MLV-based retroviral genome and a SIV envelope;
  • composition comprising a pseudotyped retroviral particle as above and/or a packaging cell line as above;
  • a method for introducing heterologous and/or homologous DNA into cells susceptible to infection by SIV env comprising infecting a cell population in vivo and in vitro with a retroviral particle as above and/or a pharmaceutical composition as above;
  • the target cell as above being a primary blood lymphocyte
  • the target cell as above being a CD4 expressing cell
  • Retroviral vector systems consist of two components:
  • the retroviral vector itself comprises a modified retroviral genome in which the genes encoding for the viral proteins have been replaced by therapeutic genes optionally including marker genes to be transferred to the target cell. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.
  • the second component is:
  • This cell line is known as the packaging cell line and consists of a cell line transfected with one or more constructs, e.g. a second or more plasmids, carrying the genes enabling the modified retroviral genome to be packaged.
  • the construct(s) direct(s) the synthesis of the necessary viral proteins required for virion production.
  • the vector is transfected into the packaging cell line.
  • the modified retroviral genome including the inserted therapeutic and optional marker genes is transcribed from the vector and packaged into the modified retroviral particles (recombinant viral particles).
  • a cell infected with such a recombinant viral particle cannot produce new viruses since no viral proteins are present in these cells.
  • the recombinat retroviral genome carrying the therapeutic and marker genes is present and these can now be expressed in the infected cell, and in all daughter cells.
  • the inventors of the present invention have established a packaging cell line packaging a MLV-based recombinant retroviral genome into a particle comprising a SIV envelope.
  • the present invention thus relates to targeted pseudotyping comprising the use of the envelope from a virus that exhibit a narrow, well defined and specific infection spectrum, namely SIV.
  • HIV-1 human immunodeficiency virus type 1
  • MLV particles MLV particles
  • the incorporation of the HIV-1 envelope into virus particles limits the virus tropism to cells expressing the CD4 marker, thus pointing to a possible in vivo utilization of such particles to target therapeutic genes especially to CD4 + cells.
  • a truncated form, but not the wild type form of HIV-1 envelope can be efficiently used for pseudotyping since pseudotypes are generated only at very low titers in the presence of a wild-type env construct.
  • SIV is a simian lentivirus closely related to HIV-1 which utilizes the CD4 molecule as a principal receptor on target cells and cause an AIDS-like disease in monkeys. Since, inter alia, primary human blood lymphocytes have been successfully transduced by SIV/MLV pseudotypes and gene transfer has been specifically restricted to the CD4 + subset the inventive pseudoptypes can be used to transduce cells which are susceptible to SIV infection and can be potentially exploited for direct in vivo delivery of gene therapy-based therapeutic approaches.
  • packaging cell line habours at least one DNA construct coding for proteins required to package the MLV-based recombinant retroviral genome. Accordingly, the core and enzymatic proteins are synthesized from at least one gag and/or pol containing construct and the envelope of SIV from the same or an independent env containing construct or even from the MLV-based recombinant genome.
  • the wild type SIV envelope may lack up to 160, especially up to 147 amino acids from the C-terminus.
  • the recombinant retroviral genome comprises preferably heterologous RNA.
  • the envelope gene of retroviruses encodes two proteins, one of which is a transmembrane protein (TM) which spans the host cell membrane and is involved in the interaction between the envelope and the core of the virus; as well as a second protein which is a surface protein (SU) which is involved in the interaction between the virus particle and the host cell during the initiation of the infection event through its anchor to the TM.
  • TM transmembrane protein
  • SU surface protein
  • the invention thus may comprise a packaging cell line as above wherein the sequence encoding the TM domain of said env containing construct has been replaced by a sequence which codes for the TM domain of MLV.
  • Suitable cell lines may be derived from rodent, human, feline or mink cells, and the gag, pol and env genes may be expressed from a promoter selected from the group of SV40, CMV, RSV, MLV or a house keeping promoter from a cellular gene.
  • the MLV-based recombinant retroviral genome is derived from a promoter conversion vector:
  • the retroviral genome consists of an RNA molecule with the structure R-U5-gag-pol-env-U3-R.
  • the U5 region is duplicated and placed at the right hand end of the generated DNA molecule, whilst the U3 region is duplicated and placed at the left hand end of the generated DNA molecule.
  • the resulting structure U3-R-U5 is called LTR (Long Terminal Repeat) and is thus identical and repeated at both ends of the DNA structure or provirus.
  • the U3 region at the left hand end of the provirus harbours the promoter. This promoter drives the synthesis of an RNA transcript initiating at the boundary between the left hand U3 and R regions and terminating at the boundary between the right hand R and U5 region.
  • RNA is packaged into retroviral particles and transported into the target cell to be infected.
  • the RNA genome is again reverse transcribed as described above.
  • a retroviral vector can be constructed in which the righthand U3 region is altered, but the normal lefthand U3 structure is maintained; the vector can be normally transcribed into RNA utilizing the normal retroviral promoter located within the left hand U3 region.
  • the generated RNA will only contain the altered righthand U3 structure.
  • this altered U3 structure will be placed at both ends of the retroviral structure.
  • any promoter including those directing tissue specific expression (see below) can be easily inserted. This promoter will then be utilized exclusively in the target cell for expression of linked genes carried by the retroviral vector. Alternatively or additionally DNA segments homologous to one or more celluar sequences can be inserted into the polylinker for the purposes of gene targeting.
  • the expression of the retroviral vector is regulated by the normal unselective retroviral promoter.
  • the therapeutic genes are expressed from a tissue specific promoter of choice introduced into the polylinker.
  • tissue specific promoter not only can virtually any tissue specific promoter be included in the system, providing for the selective targeting of a wide variety of different cell types, but additionally, following the conversion event, the structure and properties of the retroviral vector no longer resembles that of a virus.
  • Promoter conversion (Procon) vectors do not resemble retroviruses because they no longer carry U3 retroviral promoters after conversion thus reducing the possibility of genetic recombination
  • the packaging cell line thus comprises the MLV-based recombinant retroviral genome which is derived from a MLV-based retroviral vector comprising, in operable linkage, a) a 5'LTR region originating from MLV comprising the structure U3-R-U5; b) one or more coding sequences; and c) a 3'LTR region originating from MLV comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence carrying at least one unique restriction site and/or, inserted into said polylinker sequence, one or more heterologous DNA fragments, followed by the R and U5 region.
  • polylinker is used for a short stretch of artificially synthesized DNA which carries a number of unique restriction sites allowing the easy insertion of any promoter or DNA segment.
  • heterologous is used for any combination of DNA sequences that is not normally found intimately associated in nature.
  • Gene expression is regulated by promoters, in the absence of promoter function a gene will not be expressed.
  • the normal MLV retroviral promoter is fairly unselective in that it is active in most cell types. However, a number of promoters exist that show activity only in very specific cell types. Such tissue-specific or inducible promoters will be the ideal candidates for the regulation of gene expression in retroviral vectors, limiting expression of the therapeutic genes to specific target cells.
  • said heterologous DNA fragment as well as the above mentioned heterologous RNA of the retroviral genome is selected from one or more elements of the group consisting of regulatory elements and or promoters, preferably regulatory elements and/or promoters that are target cell specific in their expression, and/or regulatory elements that are regulatable by transacting molecules.
  • the target cell specific regulatory elements and promoters are preferably selected from, but not limited to, one or more elements of the group consisting of HIV, Whey Acidic Protein (WAP), Mouse Mammary Tumour Virus (MMTV), ⁇ -lactoglobulin and casein specific regulatory elements and promoters, which may be used to target human mammary tumours, pancreas specific regulatory elements and promoters including carbonic anhydrase II and ⁇ -glucokinase regulatory elements and promoters, lymphocyte specific regulatory elements and promoters including immunoglobulin and MMTV lymphocytic specific regulatory elements and promoters, and MMTV specific regulatory elements and promoters conferring responsiveness to glucocorticoid hormones or directing expression to the mammary gland, T-cell specific regulatory elements and promoters such as from the T-cell receptor gene and CD4 receptor promoter and B-cell specific regulatory elements and promoters such as immunoglobulin promoter or B29. Said regulatory elements and promoters regulate preferably the expression of
  • HIV promoter Another promoter is the HIV promoter or a minimal promoter placed under the regulation of the HIV tat responsive element (TAR) to target HIV infected cells. Targeting will be achieved because the HIV promoter is dependent upon the presence of Tat, an HIV encoded autoregulatory protein (Haseltine, W.A. 1991 , Molecular biology of the immunodeficiency virus type 1. FASEB J. 5: 2349-2360.)
  • TAR HIV tat responsive element
  • the peptide could be expressed from T cell specific promoters such as that from the CD4 or T cell receptor gene.
  • promoters from genes known to be overexpressed in these cells for example c-myc, c-fos may be used.
  • Coding sequences of the retroviral genome preferably comprises addtionally at least one non coding sequence selected from regulatory elements and/or promoters, more preferably regulatory elements and/or promoters that are target cell specific in their expression, and/or regulatory elements that are regulatable by transacting molecules, said non coding sequences regulating the expression of at least one of the coding sequences of said retroviral genome.
  • Peptide encoding sequences may be placed also under the transcriptional control of other promoters known in the art. Examples for such promoters are of the group of SV40, cytomegalovirus, Rous sarcoma virus, ⁇ -actin, HIV-LTR, MMTV-LTR, B or T cell specific and tumour specific promoters.
  • the encoded peptide may also be expressed from MMTV promoters such as the MMTV P2 promoter (G ⁇ nzburg, W. H., Heinemann, F., Wintersperger, S., Wunschhke, T., Wagner, H., Erfle, V. and Salmons, B. 1993, Nature 364, 154- 158).
  • MMTV promoters such as the MMTV P2 promoter (G ⁇ nzburg, W. H., Heinemann, F., Wintersperger, S., Miethke, T., Wagner, H., Erfle, V. and Salmons, B. 1993, Nature 364, 154- 158).
  • At least one retroviral sequence encoding for a retroviral protein involved in integration of retroviruses may be altered or at least partially deleted.
  • the MLV-based retroviral vector may contain DNA fragments homologous to one or more cellular sequences.
  • the retroviral genome especially comprises at least one coding sequence selected from one or more elements of the group comprising marker genes, therapeutic genes, antiviral genes, antitumour genes and/or cytokine genes.
  • Said marker or therapeutic gene may be selected from the group consisting of marker genes which code for proteins such as ⁇ -galactosidase, neomycin, alcohol dehydrogenase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin and secreted alkaline phosphatase, or therapeutic genes which code for proteins such as Herpes Simplex Virus thymidine kinase, cytosine deaminase, guanine phosphoribosyl transferase (gpt).
  • cytochrome P 450 and cell cycle regulatory genes which code for proteins such as SDI or tumor supressor genes which code for proteins such as p53 or antiproliferation genes which code for proteins such as melittin, cecropin or cytokines such as IL-2.
  • the packaging cell line and the pseudotyped retroviral particle according to the invention may be used for producing a pharmaceutical composition containing a therapeutically effective amount of the packaging cell line and/or of the pseudotyped retroviral particle, together with at least one pharmaceutical carrier or diluent, for somatic gene therapy in mammals including humans. Furthermore, they can be used for targeted integration in homologous cellular sequences.
  • the retroviral promoter structure is termed LTR. LTRs carry signals that allow them to integrate into the genome of the target cell. Such integrating elements can also contribute to pathogenic changes. Retroviral vectors can carry modified LTRs that no longer carry the signals required for integration. Again this increases the potential safety of these vector systems.
  • the pseudotyped retroviral particles " according to the invention can suitably be administered in the form of an injection or by implantation of the cell line according to the invention, suitably as encapsulated cells producing the pseudotyped retroviral particles according to the invention (see Danish patent application No. 740/95).
  • the Mo-MLV Gag-Pol expression construct pgag-polgpt habours the Mo-MLV gag and pol genes under the control of the Mo-MLV long terminal repeat (LTR) and a SV40 polyadenylation signal (Markowitz, D., Goff, S. and Bank, A. 1988, J. Virol. 62: 1120-1 124).
  • the construct lacks ⁇ packaging sequences as a consequence of a 134-base-pair deletion between the Mo-MLV LTR and gag gene.
  • the amphotropic Mo-MLV Env expression construct SV-A-MLVenv has the A-MLV env gene inserted between Mo-MLV LTR sequences and a SV40 polyadenylation signal ( Figure 1 B; Page, K.A., Landau, N.R. and Littman, D.R. 1990, J. Virol. 64: 5270-5276).
  • the SIV Env expression plasmid pHCMV-SIVenv ( Figure 1 B) carries the SIVmac239 env gene downstream of human cytomegalovirus early promoter/enhancer sequences (HCMV). This construct express SIV Env exploiting a Rev-independent pathway of Env expression which relies on a constitutive transport element (CTE) derived from the simian retrovirus 1 (SRV-1 ) genome
  • the Tat transducing construct pLTSN ( Figure 1 C) is a derivative of the retroviral vector pLXSN, which contains HIV-1 tat under the control of Moloney murine sarcoma virus (Mo-MSV) LTR sequences (Miller, A.D. and Rosman, G.J. 1989, BioTechniques 7: 980-990).
  • the pLXSN EGFP construct ( Figure 1 C) was used to transduce lymphoid cells ; this construct is a derivative of pLXSN carrying the gene for an enhanced green fluorescent protein (EGFP) driven by the Mo-MSV LTR (Chalfie, M. et al. 1994, Science 263: 802-805).
  • An HCMV-driven ⁇ -gal expression plasmid was used in some experiments to check for transfection efficiency.
  • 293T human kidney cells were obtained from ATCC, and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS, Gibco-BRL, Gaithersburg, MD) and 1 % L-glutamine.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • FCS Gibco-BRL, Gaithersburg, MD
  • CMMT cells expressing human CD4 and the HIV-1 LTR fused to the ⁇ -gal reporter gene
  • MAGI human HeLa-CD4-LTR- ⁇ -gal cell line
  • Lymphoid cell lines CEMX174 and C8166 were grown in RPMI1640 supplemented with 10% FCS and 1 % L-glutamine.
  • periphal blood mononuclear cells PBMC
  • PBMC periphal blood mononuclear cells
  • 293T cells were metabolically labeled for 7 h with a mixture of ( 35 S)-methionine and ( 35 S)-cysteine (Promix, Amersham, Little Chalfont, UK; 14,000 ⁇ Ci/ml, prepared in methionine and cysteine-deficient DMEM), and lysed in RIPA buffer (140 mM NaCI / 8 mM Na 2 HP0 4 / 2 mM NaH 2 P0 4 / 1 % Nonidet P-40 / 0.5% sodium deoxycholate / 0.05% SDS) as described (Mammano,
  • Pseudotyped virions were generated by transfection of 293T cells with 3 ⁇ g of the Mo-MLV Gag-Pol expression construct pgag-polgpt, along with 6 ⁇ g of either pLTSN or pLXSN EGFP as transducing vector, and 0.1 to 3 ⁇ g of the different Env expression constructs. Fresh medium was added to the cultures 12-18 h before the supernatant was collected and passaged through 0.45 ⁇ m-pore size filters.
  • CEMX174 and C8166 cell lines were performed by using either cell free supernatants or cocultivation techniques.
  • 1 ml of retroviral vector containing supernatant was incubated at 37°C with 2 x 10 5 target cells for 6-9 h in the presence of protamine sulfate (8 ⁇ g/ml) with occasional stirring. Lymphoid cells were then pelleted, resuspended in fresh medium and grown for additional 48 h before FACS analysis.
  • 10 6 cells in 1 ml RPMI 1640 medium were seeded on transfected 293T cells releasing the different retroviral vectors, and cocultivated for 9 h at 37°C. Suspension cells were then gently removed, transferred to 12-well plates containing 2 ml RPMI 1640 supplemented with
  • transduced CEMX174 cells were cultivated for 2-3 weeks in G418 containing medium (Gibco-BRL, 500 ⁇ g/ml active compound), to enrich for vector-transduced cells.
  • G418 containing medium Gibco-BRL, 500 ⁇ g/ml active compound
  • Freshly isolated human lymphocytes were transduced by cocultivation techniques, as detailed above except that, following cocultivation for 9 h, the cells were resuspended in complete RPMI supplemented with 100 U/ml recombinant IL2 (rlL2, EuroCetus, Milan, Italy), and cultured for additional 48 h prior to EGFP detection.
  • transduction of PBMC by retroviral vector-containing supernatants was carried out as described for lymphoid cell lines, but resulted in poor efficiency of gene-transfer, compared to ⁇ cultivation, and was not further pursued.
  • Neutralization assays with sera from SlV-infected monkeys were performed following a modification of a previously described protocol (Albert, J. et al. 1993, AIDS Res. Hum. Retroviruses 9: 501-504).
  • As a neutralizing serum a pooled heat-inactivated serum from 3 SlV-infected macaques was used, which contained anti-gp130 Env antibodies, as shown by prior evaluation by Western Blotting.
  • As control serum a heat-inactivated serum from an uninfected macaque was used. Briefly, 100 ⁇ l of diluted serum (six twofold dilutions, starting with the 1 :10 dilution) and 100 ⁇ l of virus- containing supernatant were mixed.
  • Vector-transduced lymphoid cells were analyzed on an Elite cytofluorometer (Coulter, Hialeah, FL). Forty-eight hours after infection cells were pelleted, washed, and fixed. CEMX174 and C8166 cells were labeled with anti-CD4-PE monoclonal antibody (mAb) (Dako, Glostrup, Denmark). PBMC were labeled with both anti-CD3ECD (Coulter, Hialeah, FL) and anti-CD8PE (Dako) mAbs. Two and three-color immunofluorescence was carried out as reported (Indraccolo et al. 1993, J. Clin Immunol. 13: 381 -388), and analyzed by using the PRISM parameter of the Elite cytofluorometer; the negative control setting for each mAb was determined by using labeled Ig of the corresponding isotype.
  • mAb monoclonal antibody
  • SIV env subgenomic constructs have been generated, expressing either wild-type (pHCMV-SIVenv) or a truncated SIV Env (pHCMV- SIV ⁇ CTenv) which lacks most of the cytoplasmic domain of the transmembrane protein.
  • this construct was generated by mutating codon 734 of SIV env gene, which also naturally undergoes mutations when SIV is passaged in human cell lines (Kodama, T. et al. 1989, J. Virol.
  • pHCMV-SIVe ⁇ v When 293T cells were transfected with pHCMV-SIVe ⁇ v, expression of precursor gp160, as well as gp130 and gp41 cleavage products has been observed. Transfection of pHCMV-SIV ⁇ CTenv resulted in expression of a smaller precursor protein, which migrated between gp160 and gp130, as well as gp130 and gp28 cleavage products. Gp28 results from the truncation of the cytoplasmic tail of gp41 , as already observed by Johnston et al. (Johnston, P.B., Dubay, J.W. and Hunter, E. 1993, J. Virol. 67: 3077-3086).
  • HCMV vector lacking env sequences was used as a negative control in the transfection experiments, and did not yield any specific band in radioimmunoprecipitation studies. Both env expression plasmids yielded comparably high levels of Env precursor glycoproteins, which were correctly processed into the expected mature products in transfected 293T cells.
  • Pseudotyped viral particles were transiently generated by cotransfection of 293T cells with the constructs shown in Figure 1 , and as detailed above.
  • a packageable transcript was provided by cotransfection of pLTSN, which expresses HIV-1 tat.
  • target cells sMAGI cell line, a macaque mammary tumor cell line which expresses human CD4 and habors a ⁇ -galactosidase ( ⁇ -gal) gene under the transcriptional control of HIV-1 LTR sequences, including the Tat responsive element (TAR) (Chackerian, B.N.,
  • A-MLV Env and of the C-terminally truncated SIV Env, respectively encoded by the SV-A-MLV-enu and the HCMV-SIV ⁇ e ⁇ v- constructs, resulted in the appearance of blue foci following ⁇ -gal staining, indicating successful vector transduction.
  • the A-MLV Env which served as a positive control, yielded titers in the order of 1 ⁇ ⁇ 5 transducing units/ml, while the truncated SIV Env generated 10- to 10Ofold less infectious viral particles.
  • the wild-type SIV Env encoded by the HCMV-SIVenv construct, could also efficiently pseudotype Mo-MLV particles, and yielded titers similar to those obtained with the truncated SIV env construct.
  • This result strinkingly differs from what has been observed in case of HIV-1 , where efficient gene transduction could be obtained only when the truncated form of the Env complex was used (Mammano, F, et al. 1997, J. Virol. 71 : 3341-3345). Positive cells were not detected when no env expression construct was used for transfection of 293T cells.
  • MAGI cells were efficiently infected by the A-MLV vector, as well as by the Mo-MLV/HIV-1 Env pseudotype (Mammano, F. et al. 1997, J. Virol. 71 : 3341 -3345).
  • Mo-MLV/SIV pseudotypes relies on gp130-CD4 interaction transduction of target cells
  • 239T cells were transfected with the gag-pol expressor along with the different env expression plasmids.
  • pLXSN EGFP which carries an enhanced form of the green fluorescent protein (EGFP) driven by the Mo-MLV LTR has been transfected.
  • Green fluorescent proteins are useful markers for gene transfer procedures, because vector-transduced target cells can be easily identified and counted by cytofluorimetrically (Chalfie, M et al. 1994, Science 263: 802-805; Zhang, G., Gurtu, V. and Kain, S.R. 1996, Biochem. Biophys. Res. Commun. 227: 707-71 1 ).
  • CEMX174 a CD4 + cell line suitable for infection by different SIV strains, including SIVmac239, has been transduced either by cocultivation or by addition of vector- containing supernatants. It has been found that transduction by cocultivation was generally a more efficient procedure, yielding up to 13.1% transduced CEMX174 cells with the amphotropic vector. Additionally, efficient infection of these cell lines by both
  • EGFP + -G418 resistant cultures could be derived from both A-MLV Env and SIV Env pseudotyped Mo-MLV particles, while no culture was obtained from the env-control. These data confirmed the results of the short-term experiments, and demonstrated the stable transduction of target cells by the Mo-MLV/SIV pseudotypes.
  • PHA-activated peripheral blood mononuclear cells from healthy donors were cocultivated with transiently transfected 293T cells; EGFP was detected on the CD4 and CD8 subsets by FACS analysis.
  • the standard A-MLV vector transduced up to 5% of CD3 + cells (range 1 -5%). Both, CD4 + and CD8 + subsets were infected at levels proportional to their percentages of each subset in the transduced population.
  • 3.4% CD3 + lymphocytes were transduced by the A-MLV vector; 2.3% belonged to the CD4 + and 1.1 % to the CD8 + subsets which accounted, respectively, for 61 ,8% and 38.2% of the total
  • CD3 + population Transduction of the cells by SIV wild-type Env pseudotype also resulted in gene transfer into the CD3 + cells with an efficiency of 0.7%. Strikingly, transduced lymphocytes belonged almost exclusively to the CD4 + subset (0.6%). Only background (0.1%) values were found among CD3 + CD8 + cells, similar to those obtained following cocultivation of lymphocytes with the env-control.

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