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EP0835320A1 - Ameliorations apportees a des vecteurs retroviraux, appropries en particulier pour la therapie genique - Google Patents

Ameliorations apportees a des vecteurs retroviraux, appropries en particulier pour la therapie genique

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Publication number
EP0835320A1
EP0835320A1 EP96915215A EP96915215A EP0835320A1 EP 0835320 A1 EP0835320 A1 EP 0835320A1 EP 96915215 A EP96915215 A EP 96915215A EP 96915215 A EP96915215 A EP 96915215A EP 0835320 A1 EP0835320 A1 EP 0835320A1
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EP
European Patent Office
Prior art keywords
gene
cells
vector
vector according
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP96915215A
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German (de)
English (en)
Inventor
Ronald Vogels
Johannes Jozephes Bernardus Boesen
Helmuth Hendrikus Gerardus Van Es
Victor Willem Van Beusechem
Domenico Valerio
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Introgene BV
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Introgene BV
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Priority to EP96915215A priority Critical patent/EP0835320A1/fr
Publication of EP0835320A1 publication Critical patent/EP0835320A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C12N2740/13023—Virus like particles [VLP]
    • 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
    • C12N2740/13041—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
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the invention relates to improved retroviral vectors which are especially useful for methods of gene therapy.
  • the invention further relates to such vectors in combination with suitable packaging cell lines, as well as virus-like particles which can be produced using said combination and methods of providing cells with genetic information of interest using said virus-like particles.
  • Retrovirus-based vectors are highly favoured tools to achieve stable integrated gene transfer of foreign genes in mammalian cells. Especially in the area of gene therapy their use has attracted considerable attention. Retrovirus-based vectors have been used in both ex vivo and in vivo gene transfer procedures and were shown to be capable of yielding long-term expression of foreign genes in culture and in vivo in animal studies as well as in man.
  • Retroviral vector systems for (safe) gene therapy purposes comprise two building blocks: the rat-ombinant- retroviral vector that carries the genetic information which is to be transduced plus all of the elements required in cis for the packaging and integration of the viral genome, and; retroviral packaging cells that provide the viral proteins encoded by the genes gag, pol and env. These polypeptides are required in trans for the production of viable virus particles but by themselves, the packaging cells are incapable of releasing infectious virus. Packaging cells transduced with the reco binant vector will therefore generate recombinant retroviruses carrying the genetic information contained in the aforementioned vector. These viruses can subsequently be used to transduce cells in which the recombinant material will be integrated following the natural retrovirus life cycle.
  • the current invention in one aspect relates to recombinant vectors with improved characteristics as compared to the previously described vectors .
  • a favoured design of the recombinant virus with properties that permit long term expression in animals and which is based on a non-pathogenic retrovirus, has previously been described (Valerio et al . , 1989; International patent application WO9307281) .
  • the gene of interest is under the transcriptional control of a viral long terminal repeat (LTR) .
  • LTR viral long terminal repeat
  • the retroviral enhancer is replaced by an enhancer active in undifferentiated cells such as embryo carcinoma (EC) cells.
  • An example is the enhancer of a polyoma virus mutant (PyFlOl) that was selected to grow on undifferentiated EC cells . This results in mutations in its enhancer that permit activity of heterologous promoters in primitive cells.
  • An LTR based on Moloney murine leukemia virus (Mo-MLV) in which the enhancer is replaced for by the PyFlOl enhancer is called ⁇ Mo+PyFlOl.
  • ⁇ Mo+PyF101-carrying vectors are superior to the commonly used vectors based on viral backbones of disease- inducing viruses such as Moloney murine leukemia virus, myeloproliferative sarcoma virus or Harvey murine sarcoma virus (Davis et al . , 1985).
  • gag open reading frame which may result in the translation of truncated viral proteins that would cause unwanted immune responses and will probably increase the potential of helper-virus formation in packaging cells carrying homologous gag sequences .
  • Sequences contained within the viral construct can function as unwanted cryptic splice sites, resulting in packaging and expression of aberrant RNA molecules, (e.g. Mclvor et al . , 1987; Sorrentino et al . , 1993).
  • the current invention discloses a basic vector and derivatives thereof that will result in improvements as related to the shortcomings stated above.
  • the current invention provides, in one aspect, a vector derived from a retrovirus, comprising a sequence responsible for transcriptional control, including an enhancer, which vector further comprises a site for insertion of at least one gene of interest, a packaging signal, said vector having no superfluous retroviral sequences and no open reading frame encoding at least parts of viral proteins, characterized in that the enhancer is an enhancer that is active in undifferentiated cells.
  • the vectors according to the invention have all the benefits of the so-called L ⁇ vectors as earlier mentioned, but they have the additional advantage that they can be used to functionally transfer material into undifferentiated cells and retaining said function throughout the differentiation and other processes.
  • the modified LTR comprising the invented enhancer will not be able to give rise to significantly pathogenic viruses.
  • the ⁇ Mo+PyF101 enhancer is a good example of such an enhancer and is a preferred embodiment of the present invention. Based on the disclosure of the present invention however, the skilled worker will be able to design other similarly suitable enhancers. In designing vectors according to the invention, the skilled worker will be able to modify the concept of the invention in order to suit his needs.
  • the ultimate goal of the vectors according to the invention lies in providing a safe and viable system of providing certain subsets of cells with additional genetic material, especially in the context of gene therapy.
  • a packaging signal is present on the vectors.
  • Such a packaging signal may be any functional one, i.e. one that works with the packaging material.
  • the packaging material will usually be provided by a cell into which the vector is transferred. Said packaging material will of course have to be functional in packaging the vectors according to the invention.
  • the most logical and most preferred combination is that of the retroviral packaging signal together with a cell that constitutively produces the retroviral proteins necessary for packaging, a so-called packaging cell line.
  • the many possible combinations of the two (kit of parts) are of course part of the present invention.
  • Both the positive mode and the negative mode of gene therapy can be realized using the vectors according to the invention.
  • the positive mode of gene therapy is intended to read upon any deficiency in a group of cells that can be treated by providing at least a number of said cells with a gene capable of removing said deficiency, such as for instance providing hematopoietic cells with a gene encoding factor VIII for correcting haemophilia.
  • the negative mode of gene therapy for this application includes the functional removal of any subset of cells within an individual by introducing genetic material into at least a number of cells from the subset. Examples are suicide genes for tumour cells.
  • the invention relates to several retroviral vectors that share a number of basic characteristics and that can be used to efficiently generate infectious recombinant virus particles when transfected into packaging cells .
  • a description of the basic embodiments present in the vectors according to this invention is given, as well as the specific characteristics of some of the preferred retroviral vectors that are included in this invention together with examples of their applications.
  • the described retroviral vectors contain a 5' LTR, preferably from Moloney murine sarcoma virus (MoMSV base 1 to
  • the remainder of the packaging signal is preferably derived from Moloney murine leukemia virus (MoMLV base 566 to 1038) with the start codon of the gag coding domain preferably mutated to a stop codon (Miller and Rosman,
  • the LTR and the extended packaging signal with the point mutations together ensure efficient packaging of the recombinant virus whithout any production of virus-derived proteins in the target cells. Furthermore, the reduced sequence homology between the MoMSV LTR and the 5' part of the packaging constructs (MoMLV LTR) generally used in packaging cells like PA317 and PG13 will reduces the chance of recombination between the constructs and thus reduces the chance of helper virus formation.
  • a splice donor site located downstream of the 5 ' LTR can in combination with the splice acceptor site just upstream of the insertion site of the gene of interest lead to enhanced translation of the inserted gene in case splicing occurs .
  • the vectors contain no overlap with the 3 ' end of the env constructs used in the above mentioned packaging cell lines.
  • Such a construct is again a preferred embodiment.
  • the resulting vector (shown in Figure 1) having all preferred features, contains a 5' and 3' LTR, a packaging signal extending into the gag coding region and a poly cloning site for the insertion of (a) gene(s) of interest and thus meets the basic requirements a retroviral vector according to the invention should meet.
  • the above basic retroviral vector is modified by deleting (parts of) the gag coding sequences without losing packaging function.
  • Such a vector further reduces the probability of recombination events in the packaging cells that may lead to replication competent retroviruses . Thus this improves the general safety features of the retroviral vectors.
  • This invention also discloses further modifications to this basic retroviral vector to allow more efficient transcription and translation.
  • a consensus Kozak sequence is introduced around the ATG of the gene of interest to improve translation of that gene.
  • the viral enhancer in the 3' LTR is replaced by a mutant form of the Polyoma virus enhancer that is specifically selected for activity in F9 embryonal carcinoma cells (Linney et al . , 1984) and that is known to be less sensitive for promoter inactivation in haemopoietic stem cells and early haemopoietic progenitor cells compared to the wild type MoMLV enhancer (Valerio et al . , 1989; Van Beusechem et al . , 1990).
  • Another advantage arising from the replacement of the wild type MoMLV enhancer for the polyoma enhancer is that the resulting ⁇ Mo+PyF101 LTR renders murine leukemia viruses into non-pathogenic viruses (Davis et al . , 1985). Following one round of replication this alteration contained within the U3 region of the 3 ' LTR is also tranferred to the 5' LTR and is thus present in both LTR's of the proviral sequence. As a consequence, after infection of an amphotropic packaging cell line, the ⁇ Mo+PyF101 LTRs contain less sequence homology with the packaging constructs as compared to LTRs with wtMoLV enhancers. Additional modifications to improve expression in target cells include:
  • LCR Locus Control Region
  • Selection genes include the neomycine r gene, the hygromycine r gene, a gene encoding a fluorescent protein or a gene coding for a biologically inactive transmembrane molecule that all allow for efficient selection in vitro .
  • selection markers may be included that also allow for selection of transgene expression in vivo like for example, without limitation, the human genes for Multi Drug Resistance (MDR-1 see examples 2 and 3), a UDP-Glucuronosyl Transferase, Thymidylate Synthetase, canalicular Multispecific Organic Anion Transporter, ⁇ -Glutamyl Cysteine Synthetase, as well as biologically active mutants of these genes.
  • MDR-1 Multi Drug Resistance
  • - additional (regulatory) sequences that can influence the expression of the integrated provirus in the target cells e.g. boundary elements and/or (tissue-specific) promoters or enhancers.
  • Preferred or additional properties of the retroviral vectors described in this invention include:
  • a dicistronic transcription unit whereby the two coding regions are separated by a short non-coding linker allowing efficient reinitiation of the ribosomal complex on the start codon of the second gene.
  • This non-coding linker can have a variable length but is devoid of any ATG sequences or sequences that form strong secondairy structures in the RNA.
  • the length of a favourable intercistron is a multiplicity of 3, the stop codon of the first gene thus placed in frame with the start codon of the second gene.
  • the retroviral vector pLXSN (Miller and Ros an, 1989) was digested with Nhel and the insert containing the viral sequences was ligated into the vector backbone pUC19 obtained from pSFG- tpa ( R. Mulligan and I. Riviere, hitehead Institute for Biomedical Research, Cambridge, MA) after digestion with Nhel.
  • the resulting construct named pLXSNi9, was digested with BamHI and the ends were filled in using the Klenow enzyme. After removal of the enzymes, the DNA was digested with Nhel after which the 1452 bp Nhel/BamHI fragment containing the 5' LTR and the extended packaging signal was isolated.
  • a 98 bp fragment containing the 3' LTR was isolated from the vector pLNSX (Miller and Rosman, 1989) following digestion with Nhel and StuI. Ligation of these fragments into the Nhel fragment from pSFG-tpa containing the pUC19 backbone resulted in the viral construct pLec ( Figure 1) .
  • pSK/Zip ⁇ Mo-PyFlOl has been generated by subcloning of the Clal-EcoRI fragment from pZip ⁇ Mo- ⁇ -PyF101(N " ) into the pBluescript vector (M. Einerhand, TNO, Radiobiological Institute).
  • pZip ⁇ Mo+PyF101(N " ) is a low copy vector (pBR 322 based) containing a 3' LTR that has been made by combining the Clal/Kpnl fragment from pMLV- C/R/B ⁇ Mo+PyF101 (Linney et al .
  • the first contains part of the R region and a U3 region in which the enhancer sequences have been replaced by a mutant form (F101) of the Polyoma virus enhancer
  • the- second contains the remaining R and U5 sequences of the 3' LTR.
  • Example 1 Monocistronic and bicistronic retroviral vectors for suicide gene therapy
  • Suicide genes like the Herpes simplex virus type 1 thymidine kinase (HSV-tk) gene or the cytosine deaminase gene, encode proteins that are capable of transferring a non-toxic prodrug into a toxic drug.
  • HSV-tk Herpes simplex virus type 1 thymidine kinase
  • cytosine deaminase gene encode proteins that are capable of transferring a non-toxic prodrug into a toxic drug.
  • the prodrug ganciclovir is not toxic for eukaryotic cells but after (mono)phosphorylation by the HSV-tk gene it will be converted into a nucleotide analog by cellular enzymes. Incorporation of this analog into the DNA of replicating cells results in chain termination and cell death.
  • tumour models in rats to show that after transfer of a suicide gene into tumour cells in vivo, only a minority of the tumour cells need to express the suicide gene in order to establish an effective anti-tumour respons (reviewed in Moolten, 1994).
  • Efficient transduction of tumour cells can be achieved in vivo by direct injection of retrovirus producer cells into the tumour (Culver et al . , 1992) .
  • retroviral vectors outlined in this invention we describe the vector pIGTk and its use in suicide gene therapy for malignant brain tumours in a rat model.
  • pIGTk The retroviral vector pLec was digested with Xhol, blunted with Klenow and subsequently digested with BamHI. After dephosphorylation using calf intestine phosphatase (CIAP), this fragment was ligated to a fragment containing the coding region of the Herpes simplex virus type 1 thymidine kinase gene (HSV-1 tk) obtained from pAdTK (Bram Bout, IntroGene; European Patent Application 94202322.7) by digestion with HindiII, blunting with Klenow and digestion with BamHI. The resulting viral vector was named pLTk. pLTk was linearised with Bsi I, partially digested with
  • a retroviral vector was created that only contains the coding sequences of the HSV-1 tk gene with an optimized Kozak sequence around the start codon.
  • the Ncol site 5' and the BamHI, Avrll, Hindlll and Clal sites 3' of the inserted gene are useful cloning sites to swap inserts in this vector ( Figure 4a) .
  • the Nhel fragment from pLTKkoz was introduced into the unique Nhel site from pSK/Zip ⁇ Mo+(PyFlOl) to generate construct pIGTk ( Figure 4b) .
  • the retroviral vector pIGTk was cotransfected into the amphotropic packaging cell line PA317 (ATTC No. CRL 9078) together with an expression construct containing the neomycine r gene ( ⁇ Mo+PyF101LTR-Neo, M. Einerhand unpublished) and G418 resistant clones were isolated.
  • neomycine r gene ⁇ Mo+PyF101LTR-Neo, M. Einerhand unpublished
  • G418 resistant clones were isolated.
  • One of these produced around lxlO 5 infectious virus particles/ml that were capable of transferring the HSV-1 tk gene to thymidine kinase deficient Rat-2 cells.
  • This virus producer (termed IG-RV-TK) has been successivefully used in a preclinical study aimed at curing experimental brain tumours in rats (Vincent et al . , 1996).
  • FIG. 5 clearly shows the prolonged survival of rats treated with IG-RV-TK producer cells in combination with ganciclovir (Vincent et al . , 1996) .
  • the tk/ganciclovir system can also be of great value in the treatment of leukemia.
  • patients treated for leukemia often receive an allogeneic bone marrow transplantation (BMT) , using a BM graft from an (un)related, but closely MHC-matched donor.
  • BMT bone marrow transplantation
  • T-cells in such a graft has turned out to be a major factor determining the successive of the treatment.
  • a solution to this problem could be to isolate (allogeneic) peripheral blood lymphocytes from the donor prior to the BMT, transduce them in vitro with a suicide gene and use these cells together with the T-cell depleted BM graft (Tiberghien et al . , 1994).
  • GVHD develops treatment of the patients with ganciclovir will result in selective killing of the activated (transduced) T-cells prospectively leading to abrogation of GVHD.
  • patients may still benefit from a GVL reaction resulting in a decreased rate of leukemia relapse.
  • both genes are located in a dicistron driven by one (viral) promoter.
  • Dicistronic mRNAs allow for efficient translation of the second gene if the genes are separated by either a short intercistronic linker depending on ribosome scanning, or by specialised sequences triggering internal binding of ribosomes .
  • the small ribosomal subunit binds to the 5' end of the capped mRNA and scans for the presence of ATG sequences. Initiation of translation occurs at the first ATG in a favourable context (Kozak, 1987b) and the translation complex dissociates when a stop codon is encountered.
  • a favourable dicistronic mRNA we describe here the construct pLTk+Neo ⁇ Mo in which the tk gene and the neo r gene are separated by a 36 nucleotide linker.
  • the tk or the neo r gene can both be replaced by other genes.
  • a favourable intercistronic linker (1) Has the start codon of the second gene inserted in frame with the stop codon of the first gene; the length thus being a multiplicity of 3, (2) should not contain any ATG sequences, (3) can vary in length between 9 and 200 bp and (4) should not contain sequences that form strong secondary structures in the RNA.
  • the two genes with their intercistrons can subsequently be introduced into the retroviral construct. Const-ruction of pLTk+ ⁇ eo ⁇ Mo
  • the HSV-tk gene was excised from pLTKkoz using EcoRI and BamHI and subcloned into pUC119.
  • An EcoRI/HincII fragment containing the promoter of the human phosphoglycerate kinase gene (Singer-Sam et al . , 1984; Michelson et al . , 1983) was provided with an EcoRI linker and introduced into the EcoRI site generating pPGK-Tk.
  • neo r gene containing the 3' part of the neo r gene was then introduced into this vector after digestion with Clal followed by dephosphorylation and partial PstI digestion.
  • the modified neo r gene was isolated as a BamHI/Sall fragment and cloned into the corresponding sites in pPGK-TK, resulting in pPTk+ ⁇ eo/IF.
  • pAMG-1 Valerio et al . , 1985
  • HBV hepatitisB virus
  • the stop codon of the tk gene is positioned in frame with the start codon of the neo r gene.
  • a second Tk- ⁇ eo dicistron has been generated in which the two coding regions are separated by a 35 nucleotide linker in stead of 36 nucleotides (pPTK- ⁇ eopA/OF; Figure 6).
  • Tk+ ⁇ eo dicistron from pPTk-r ⁇ eopA/IF was excised by EcoRI and Sail digestion and ligated into the pLec vector after digestion with EcoRI (partial) and Xhol.
  • pLTk+ ⁇ eo ⁇ Mo was completed after subcloning of the ⁇ hel fragment into the corresponding site in pZip ⁇ Mo+PyF101( ⁇ " ) Efficient translation of the second gene in a bicistron
  • Two monocistronic (control) constructs were generated carrying either a single TK coding region (pPTkpA) or the neo r coding region (pPNeopA; Figure 6).
  • plasmid DNA (16 ⁇ g) was cotransfected with 4 ⁇ g pCMVLuc (L. Fortunati and M.
  • Table 1 Relative number of colonies obtained after transfection of the different constructs into Rat-2 cells grown in indicated selective medium. Each transfection has been performed in duplo. See text for details.
  • the G418 selected pools containing the constructs pPTk ⁇ NeopA/IF, pPTk-NeopA/OF or pPNeopA and the HAT selected pool pPTkpA can be used to test the sensitivity of the cells to the prodrug ganciclovir. To test this, cells were plated at low density (lxlO 4 cells) in 75 cm 2 flasks in the presence of 5 ⁇ M
  • Ganciclovir Ganciclovir
  • pPTkpolioNeo contains an IRES sequence derived from poliovirus and the second, pPTkemcvNeo containes an IRES sequence from EMCV (encephalomyocarditis virus).
  • EMCV encephalomyocarditis virus
  • Hindlll-EcoRV fragment from pP2-5' (Pelletier et al . , 1988) into pPTk+NeopA/IF digested with Xbal followed by Klenow treatment.
  • the resulting construct was then digested with BamHI and partially digested with Ncol, Klenow treated and religated to remove most of the linker sequences present in pPTk ⁇ NeopA.
  • pPTkemcvNeo was generated by inserting a 582 bp blunt EcoRI, MscI fragment from pBS-ECAT (Jang et al . , 1989) into a pBr322 based pPTk ⁇ NeopA clone.
  • the resulting vector was modified by exchanging the sequences between the Hindlll site in the IRES sequence and the Ncol site at the 5' end of the neo coding sequences for a fragment from the 3 ' end of the EMCV IRES with the sequences around the translation start site modified to an Ncol site thus placing the ATG in the neo gene onto the starting ATG from the EMCV IRES.
  • the EMCV IRES with a Ncol site on the ATG has been made by exchanging the 3' end of an EMCV IRES clone in pUC119 for a per fragment generated with the following primers: 5 ⁇ -CCCAGTGCCACGTTGTGAGTTGG-3 ' and 5' -GCGGATCCGGCCATGGTATCATCGTGTTTTTC-3 * .
  • Rat-2 fibroblasts were cotransfected as described above with pRSVLuc and one of the following bicistronic constructs : pPTk+ ⁇ eopA/IF, pPTk- ⁇ eopA/OF, pPTkpolioNeo, pPTkemcvNeo, pPTkpA or pPNeopA ( Figure 6).
  • Table 2 % of surviving cells grown in medium with different concentrations GCV relative to cells grown in normal medium
  • HSV-tk + cells showed that, at the cell densities used in these experiments, there is no influence of the so called 'bystander effect', proving that cell kill is due to endogenous expression of HSV-tk and not due to transfer of the toxic substance to tk " cells (results not shown) .
  • intercistronic linkers can be used in a bicistronic transcription unit in st.ead of IRES sequences without losing functional expression of either gene in the bicistron. Synthetic intercistronic linkers are preferred over virus-derived IRES sequences because they are shorter in size and do not form strong secondary structures in the RNA.
  • Multidrug resistance may result from synthesis of a multidrug transporter (P-glycoprotein) encoded by the 'multidrug resistance (MDRl ) ' gene. It is possible to confer a multidrug-resistant phenotype to drug-sensitive cells by transfection and subsequent expression of the MDRl gene. An attractive approach therefore would be to introduce the MDRl gene into haemopoietic stem cells (HSC) , with the objective to protect patients from drug-induced myelotoxicit . Alternatively, the MDRl vector could be used to introduce yet another gene of interest (e.g.
  • Glueocerebrosidase gene or HIV inhibiting genes/sequences allowing selection in vivo, using cytotoxic drugs efficiently pumped out of the cell by P- glycoprotein.
  • Successful circumvention of myelosuppression by transduction of the MDRl gene in bone marrow cells is dependent on an efficient gene transfer system.
  • retrovirus-mediated gene transfer is the only technique that allows efficient and stable gene transfer into HSC.
  • An optimal retroviral construct for introduction of the human MDRl gene in haemopoietic cells has besides the properties of the basic pLec ⁇ Mo construct the following additional properties:
  • mutant MDRl P-glycoproteins may result in vivo in an immunogenic response to transduced cells.
  • the high-copy number plasmid backbone in pLTKkoz was replaced by a low-copy number plasmid by subcloning of the Nhel fragment of pLTKkoz in pZIP ⁇ Mo+PyF101(N-) .
  • the resulting construct was named LTK- ⁇ Mo.
  • the wild-type human MDRl cDNA van der Bliek et al . , 1988
  • Fragment 1 NcoI-BamHI fragment of LTK- ⁇ Mo.
  • Fragment 2 Ncol-EcoRI MDRl fragment.
  • This 1178 bp fragment was generated by PCR using Pfu DNA polymerase.
  • Two primers were used: ' • mdr5' (thio) ' (5'- CCTCTAGACCATGGATCTTGAAGGGGACCGCAA TGGAGGA-3') spanning the start-codon of MDRl , in which a cytosine was placed before the ATG start-codon, thereby creating a Ncol site at this position.
  • the second primer (4728A: 5' -CCAACCAGGGCCACCGTCTGCCCA-3' ) is positioned 3' of the EcoRI site in MDRl. This fragment was first digested with EcoRI and subsequently partially digested with Ncol. The 1.2 kbp fragment was isolated from an agarose gel . Fragment 3: EcoRI-BamHI 3' MDRl fragment.
  • This fragment was ligated to two double strand linkers: a 5' -linker (5'- CTCTGAGCTCCCATGGATCTTGAAGGGGACCGCAATGGAGGAGCAAAGAAGAAGAACTTTTT TAAATCTC-3 ' ) which was cut with Ncol and Dral and isolated from a agarose gel and a 3'-linker (5'-
  • IGmdrl-G was transfected in the PA317 packaging cell line (obtained from the American Type Culture Collection: ATCC No. CRL 9078).
  • PA317 cells were selected in HAT medium to select for cells that retain the packaging function. The cells were grown in HT medium for 4 days to dilute residual amethopterin. 6xl0 5 cells were transfected with 6 ⁇ g IGmdrl-G DNA using LipofectAMINE as reagent. The following day cells were trypsinized and l.OxlO 6 cells were seeded per 75 cm 2 dish. The next day 70 nM of vincristine was added. Medium with vincristine was refreshed every 3 days. Two weeks after trypsinization vincristine resistant colonies were trypsinized, pooled, and further cultured. The resulting cell line was called IGvpOlO.
  • IGmdrl-G Transduo+ion and expression studies with IGmdrl-G a) Test for the presence of helpervirus IGvpOlO was proven to be free of replication competent retroviruses (RCR) . This was determined by co-cultivation of 5xl0 6 cells for 5 passages with Mus dunni cells which permits amplification of RCR by the feline (PG-4) S+L- focus assay. b) Expression of IGmdrl-G in a human drug-sensitive A2780 cell line.
  • CD34" r selected PBPC were transduced over 96 hours with IGvpOlO supernatant at a ceil concentration of 1 x 10 6 /ml in the presence of human interieukin-3. IGvpOlO supernatant was refreshed every 24 hours. Protamine sulphate (4 ⁇ g/ml) was added with every supernatant change.
  • MDRl-transduced and mock- transduced PBPC were plated in duplicate at 5 x 10 3 /ml in 1 ml methylcellulose medium in the presence of IL-3 and GM-CSF. Screening for MDRl overexpressing progenitor cells was performed with vincristine which is an efficient substrate for the P-glycoprotein.
  • CD34 + selected normal human bone marrow cells were transduced as described for PBPC. After transduction, cells were seeded for CFU-C formation in the presence of increasing amounts of vincristine. Individual CFU-Cs were picked and D ⁇ A isolated from the colonies was subjected to a provirus- specific PCR. Seven independent experiments demonstrated that 8 ⁇ 9 percent of the CFU-C was transduced with the IGmdrl-G retrovirus. From one experiment, also vincristine resistant colonies were analyzed. This experiment showed that the percentage PCR+ CFU-C increased from 30% without vincristine to 44% (20 nM) , 71% (30 nM) and 100% at 40 nM drug. This experiment clearly demonstrates that in vitro selection of transduced hemopoietic progenitor cells at increasing doses of cytostatic drug actually occurs.
  • Example 3 Gene therapy for AIDS/ ⁇ n vivo selection of transduced HSC Infection of CD4- T-cells by the human immunodeficiency virus (HIV) is the first and causative event in the development of AIDS.
  • HIV human immunodeficiency virus
  • Gene therapy strategies have been developed that interfere with the life cycle of the retrovirus using so called genetic antivirals like e.g. intracellular antibodies, ribozymes, antisense molecules or decoys (reviewed in Gilboa and Smith, 1994).
  • haemopoietic stem cells are the targets for such protective therapy as they will provide the patient with a continuous source of protected T-cells.
  • the stable infection of HSC may not be very efficient and following transplantation of the transduced cells a multitude of non- transduced endogenous stem cells will continue to generate mature cells resulting in many unprotected cells in the peripheral blood and thus facilitating replication of HIV.
  • the constructs that we describe here and that are designed to express genetic antivirals in HSC and descendents thereof, are all based on the pIGmdrl-G retroviral construct (see example 2).
  • the human MDR-1 gene allows for selection of transduced stem cells in vivo.
  • retroviral constructs generating antisense RNAs directed to the 5' end of HIV-1.
  • the use of the polymerase Ill-dependent adenoviral VA1 promoter ensures high levels of expression of short inserted sequences.
  • pIGmdrl-G a subclone from pIGmdrl-G was generated by digestion with BamHI and religation of vector sequences.
  • This clone, pIGmdr ⁇ BamHI was used to introduce the Adenoviral VA1 gene and promoter sequences that were obtained by amplification of Ad5 sequences with the primers: 5' -CCTGCTAGCTCTAGACCGTGCAAAA- 3 ' and 5 * -AAAGCTAGCAAAAAAGCGGCCGCGGGGCTCGAACCCCGGTCGTCC-3 ' .
  • Digestion of the per product with Nhel allowed for cloning into either the unique Avrll or Nhel site of pIGmdr ⁇ BamHI.
  • Clones were selected that contained the VA1 promoter in either orientation.
  • These were obtained by amplification of HIV-1 sequences in the pBRU2 vector (Xbal/Clal fragment from pLAI/pBru from B.Klaver, AMC A' dam) using the per primers: 5' TAR 5 ' -AATCGCGGCCGCGTCTCTCTGGTTAGAC-3 ' with 3' TAR 5' -AATCGCGGCCGCGGTTCCCTAGCTAGCC-3' to amplify the TAR loop from +1 to+57 (pIGmdrl-G/HIV-TAR) and with 3' gag 5'- AATCGCGGCCGCTCTCGCACCCAT-3 ' to amplify the 5' end up to the gag start codon from +1 to +348 (pIGmdrl-G/HIV-gag) .
  • the per fragments were digested with NotI and cloned into the pIGmdr ⁇ BamHI/VA constructs.
  • all constructs were derived in two orientations: one in which the VA1 promoter is driving transcription in the same direction as the viral LTR and one in the reversed direction.
  • Retroviral constructs for the treatment of Gaucher disease should be based on vectors working favourably in the haematopoietic system particular following stem cell gene tranfer.
  • the construction of IGGC therefor carries the glueocerebrosidase (GC) sequence in the retroviral back bone as disclosed in this invention.
  • IG-GC retroviral vectors that differ in the length of the inserted Glueocerebrosidase (GC) cDNA (described below) was undertaken in order to perform in vivo gene marking studies.
  • the difference in length of the inserted GC cDNA's allows for the discrimination between multiple retroviruses after ex vivo infection and reinfusion of the infected graft into the same animal or human.
  • these vectors can be used to study transduction efficiency differences between viruses produced by different packaging cell lines.
  • different transduction protocols i.e. different growth factors and the role of virus titers can be studied.
  • gene marking with multiple, distinguishable vectors enables one to rapidly assess the role of crucial parameters in determining transduction efficiency of CD34 * primitive progenitor cells.
  • an additional advantage of the use of therapeutic cDNA's, such as hGC, in gene marking studies instead of cDNA's encoding foreign proteins, for instance Neo r (Brenner et al . , 1993), is the absence of unwanted host immune responses against the expressed foreign protein.
  • the complete cDNA sequence (1888 bp) of the human placental glueocerebrosidase was digested to completion by Xhol and separated from the 7549 bp pGB125 backbone (Genzyme cooperation) by agarose gel electrophoresis.
  • the DNA fragment was electroeluted from the agarose and purified by phenol/chloroform/ isoamylalcohol extraction.
  • the pLec plasmid (5773 bp) was linearized by Xhol digestion.
  • the Xhol digested DNA was treated with calf intestinal phosphatase (CIAP) and subjected to agarose gel electrophoresis.
  • the linearized DNA fragment was excised and purified.
  • the isolated Xhol hGC cDNA fragment of 1888 bp was ligated to the dephosphorylated 5773 kb Xhol DNA fragment of pLec using T4-DNA ligase.
  • the resulting 7661 bp retroviral vector is designated IG-GC-1 ( Figure 9).
  • the IG-GC-1 vector was digested with Nhel.
  • the resulting 3431 bp Nhel DNA fragment was ligated to the linearized and dephosphorylated 4375 kb Nhel DNA fragment of pSK/Zip ⁇ Mo+PyFlOl using T4-DNA ligase.
  • the resulting 7806 bp retroviral DNA construct is designated IG- GC-2 ( Figure 10) .
  • IG-GC-3 and IG-GC-4 two variants were constructed designated IG-GC-3 and IG-GC-4. These variants are identical to IG-GC-1 and IG-GC-2 respectively except for the 3' -untranslated region of the hGC coding sequence. From this region a 160 bp fragment (from nt. 1728 to nt. 1888) was deleted using PCR. Construction of IG-GC-3 was done as shown in figure 11. Briefly, two oligonucleotides were synthesized, GCo3 with sequence 5' -CGGGATCCTAGAGGGGAAAGTGAG-3' and GCo4 with sequence 5' -CAGCCCATGTTCTACCAC-3' . GCo3 contains a BamHI restriction site.
  • IG-GC-2 oligonucleotide
  • the 420 bp PCR fragment was digested with BamHI and the 220 bp PCR fragment was isolated and ligated to IG-GC-1 DNA that was linearized with BamHI ( Figure 11) .
  • the resulting vector, designated IG-GC-3 now contains a human GC cDNA which lacks 160 bp in the 3' noncoding region ( Figure 12).
  • IG-GC-4 IG-GC-3 was digested with Nhel and a 3231 bp DNA fragment was isolated.
  • IG-GC-2 and IG-GC-4 plasmid DNAs were introduced into ecotropic retroviral packaging cell lines GP * E86 (Markowitz et al . , 1988) and ⁇ -CRE (Danos et al . , 1988) respectively. Selection of transfected cells was achieved by cotransfection of expression plasmids pSV2neo (Southern and Berg, 1982) with ICG-GC-2 and pPGKneo (R. Vogels, see example 1) with IG-GC4 at a ratio of 1 : 10. G418 resistant cell pools GP2b and ⁇ 4c were generated.
  • Ecotropic IG-GC-2 and IG-GC-4 virus was produced by growing confluent layers of ecotropic producer cells in fresh medium at 32 °C for 24 hours. Virus containing supernatants were collected, passed through a 0.45 ⁇ m filter, aliquoted and immediately frozen in liquid nitrogen followed by storage at -80 °C. To test whether the G418 resistant ecotropic cell pools also express the human GC protein, cell lysates were made and the GC enzyme activity level was measured using an artificial GC substrate (fluorescence: 4-Mu- ⁇ -glucoside or colorimetric: PNP- ⁇ -glucoside) .
  • 3T3 cells had 1.8-2.0 times elevated GC activity levels compared to the non-transfected packaging cells ( Figure 14A) .
  • a Western blot of cell pools A, B and C (3 independent IG-GC-2 transfeetions) using the human GC specific monoclonal antibody 8E4 (Aerts et al . , 1985) showed that the 59 kDa hGC protein is expressed in the GP ⁇ E86 packaging cell line ( Figure 14B) .
  • ecotropic virus obtained from the GP2b pool was used to multiply infect amphotropic PA317 cells.
  • PG13 Gibbon ape leukaemia virus packaging cells
  • hGC activity was measured as described above and again proved to be 2 to 3 times elevated compared to parental PG13 cells (data not shown) .
  • producer clones were isolated from these pools. In order to achieve this two rounds of limiting dilution ( ⁇ 1 cell/well) were performed in 96-well microtiter plates. Virus production from these clones was initially tested by measuring hGC activity in NIH/3T3 cells (PA317 derived) or Rat-2 fibroblasts (PG13 derived) after incubation with the cell culture supernatant. Clones giving the highest increase in hGC activity were selected and designated
  • PA2 PA3 17/IG-GC2
  • PA4 PA3 17/IG-GC4
  • PG2 PA13/IG-GC2
  • PG4 PG13/IG-GC4
  • lysates were prepared from the producer cells.
  • lysates were prepared from Gaucher type II fibroblasts infected with PA2 , PA4 , PG2 , and PG4 virus supernatants to compare virus titer.
  • a Western analysis of these protein lysates indicates a correlation between endogenous hGC expression in the producer clones and virus titer i.e. the producers with high endogenous hGC levels yield high titers (Fig. 16).
  • the virus titer of the PA2 producer was determined by incubating 10 5 ⁇ IH/3T3 cells (6-well plates) with 1 ml of PA2 virus supernatant over a 48 hour period. Subsequently, the infected NIH/3T3 cells were subjected to one round of limiting dilution (>1 cell/well) and 50 individual clones were expanded in order to measure hGC activity and to isolate genomic DNA for copy number determination. All of these 50 NIH/3T3 clones expressed hGC. Southern analysis revealed that in each transduced individual NIH/3T3 clone at least three separate integrations took place. From these results it was concluded that the titer of the PA2 producer is at least 3 x 10 5 functional virus particles/ml. (Fig. 17).
  • CD34+ cells were isolated from total bone marrow- harvested from a Gaucher type I patient. These CD34 ⁇ cells were seeded at a concentration of 10 5 cells in 24-well plates in 400 ml virus supernatant supplemented with 4 mg/ml protamine sulphate, IL-3, and pen/strep. Daily, for four executive days, the virus supernatant was refreshed. After this transduction period 2.5 x 10 4 cells were seeded in a liquid culture assay (medium containing IL-3, IL-6, SCF, GM-CSF, and G-CSF) . After a 10 day incubation at 37°C/10% CO2 the cells were harvested and counted.
  • a liquid culture assay medium containing IL-3, IL-6, SCF, GM-CSF, and G-CSF
  • the number of cells obtained after this 10 day period normally was between 5 x 10 5 -1 x 10 6 showing a proliferative capacity factor of 20 to 40 times.
  • Nine-tenth of the cells was used to measure elevation of hGC activity.
  • One-tenth of the cells was pelleted and lysed for PCR.
  • Figure 19 shows the elevation of hGC activity in the differentiated Gaucher type I cells derived from the liquid culture after transduction with either IG-GC2/IG-GC4 or MDR virus.
  • PCR and subsequent Southern analysis of the cells derived from the liquid culture shows that the provirus is present in the IG-GC2/IG-GC4 transduced cells (fig. 20).
  • CD34 + cells were also seeded for a colony forming unit (CFU) assay at a concentration of
  • the IG-derived viruses are potent gene delivery vehicles capable of correcting the biochemical Gaucher phenotype in primary fibroblasts and CD34 "r he opoietic Gaucher cells.
  • methotrexate resistant cDNA of human dihydrofolate reductase hDHFR
  • the human wild type DHFR was amplified with Pwo enzyme from single stranded cDNA synthesized from mRNA of human liver.
  • the 5 'oligonucleotides DHFR1 The 5 'oligonucleotides DHFR1
  • IG-GC2 plasmid was utilized for the amplification of a 300 bp hGC DNA fragment with oligonucleotides GColl (5 1 -gatcgagggatgcagtac-3 ' ) and oligonucleotide GCol4 (5' -tgtggcgtcgccagtgaggatcctctagaagcttggg-3 ' ) .
  • Oligonucleotide GCol4 contains the stopcodon of hGC. Downstream of oligonucleotide GColl a unique Sail site is present and oligonucleotide GCol4 contains a Hindlll site.
  • the wildtype and Phe32Ser mutant hDHFR PCR fragments were digested with Hindlll (present in DHFR1) and Clal (present in DHFR2) .
  • the 300 bp 3 ' -hGC PCR fragment was digested with Sail and Hindlll. These two DNA fragments were ligated in a three point ligation reaction into a Sail, Clal digested IG-GCl construct (Fig. 22).
  • the resulting bicistronic vectors were coded IG-GC5 (wildtype hDHFR) and IG-GC6 (Phe32Ser hDHFR) .
  • the hGC coding region is separated from the hDHFR coding region by a 36 bp intercistronic linker enabling translation of both proteins from one single mR ⁇ A.
  • the ⁇ hel fragments were isolated and cloned into pSK/ZipDMo+PyFlOl (IG-GC7 and IG-GC8 respectivily) .
  • bicistronic retroviral vectors can be transfected into retroviral packaging cell lines to generate viruses that upon infection of target cells render these cells resistant to methotrexate, a potent cytotoxic drug which inhibits D ⁇ A synthesis by depleting the pool of pyrimidines.
  • Example 5 Introduction of Locus Control Region sequences of the human CD2 gene in the recombinant retroviral vector pLg * AL( ⁇ Mo+PyF101) .
  • the retroviral vector construct pLgAL( ⁇ Mo+PyF101) (Van Beusechem et al . , 1990) was used, wherein A represents the human cD A gene encoding adenosine deaminase (hADA) , which is further referred to as "the vector”.
  • the Locus Control Region (LCR) sequence from the 3 'regionof the human CD2 gene (Lang et al . , 1988) was used, which is further referred to as "CD2-LCR”.
  • nt 2-2077 a 2076 nt Hindlll fragment (nt 2-2077) has been identified which in transgenic mice exerts all the characteristic features of the CD2-LCR on the CD2 promotor as well as on heterologeous promoters (Lang et al . , 1988, Lang et al . , 1991). Within this fragment lies a 880 nt Afllll fragment (nt 433-1314), of which it has been shown in human T-cell lines in vitro that it act as a CD2-LCR (Lake et al . , 1990).
  • the Hindlll CD2-LCR fragment further referred to as "L2”
  • the Afllll CD2-LCR fragment further referred to as "L0.8”
  • L2 and L0.8 fragments were isolated from the construct GSE1502 (D. Kiousis, MRC) and provided with a blunt end with Klenow-polymerase.
  • the vector was digested with Clal (nt 7675 of Mo-MuLV, in env) or ⁇ hel (nt 7846 of Mo-MuLV, in the 3 'LTR) and also provided with a blunt end.
  • the 8 new constructs were packaged into recombinant retroviruses. Thereto 20 ⁇ g DNA of the constructs was transfected into the ecotropic packaging cell line GP+E-86 (Markowitz et al . , 1988), using the method as described by Chen and Okayama (1977). Prior to the transfection the GP+E-86 cells were cultured in a medium containing 15 ⁇ g/ml hypoxanthine, 250 ⁇ g/ml xanhine, and 25 ⁇ g/ml mycophenolic acid, in order to select for retaining the DNA sequences which are responsible for the production of viral proteins.
  • Transfectants producing a functional hADA enzym were isolated through a selective culture in medium containing 4 ⁇ M xylofuranosyl-adenine (Xyl-A) and 10 nM deoxycoformycin (dCF) (Kaufman et al . , 1986).
  • Culture supernatant of Xyl-A/dCF- resistant transfectants was, after filtration through a filter with a pore size of 0.45 ⁇ m, used to transduce the amphotropic packaging cell line GP+envAml2 (Markowitz et al, 1988) with the ecotropic recombinant retroviruses present in that culture supernatant.
  • amphotropic packaging cells were selected for retaining the DNA sequences encoding viral proteins prior to use (as described for GP- * -E-86 cells, with the addition of 200 ⁇ g/ml hygromycine B) and preincubated with 4 ⁇ g/ml polybrene to promote retrovirus transduction.
  • GP+env Aml2 cells producing a functional hADA enzym were isolated through Xyl-A/dCF-selection as described above. Individual hADA- positive GP+e._vAml2 clones were isolated and expanded.
  • NL2R clones harbouring truncated retrovirus insertions was subjected to a more thorough analysis.
  • Three independent NL2R clones were all shown to have a 2 kb deletion in a fragment covering the area from the 3 'end of the hADA gene until the 3 'end of the L2R insertion.
  • NL2F clones the retrovirus insertions were shown to have deletions varying from 200 bp to 2 kb in size.
  • the deletion comprised (a part of) the L2F fragment, in 2 of these clones the deletion extended into the vector (3 ' of hADA and 5' of L2F in the 3 'LTR).
  • the 5 damaged CL0.8 clones had deletions of different lengths in 4 cases and an insertion in one case.
  • the L2 fragment comprises sequences which influence the stability of the vector negatively when this fragment is incorporated the LTR of the vector. Therefore, L2 fragments must be placed between the LTRs, whereby it is preferred to use the Clal site for that purpose.
  • the smaller LO.8 fragment can be used as an alternative for insertion into the LTR.
  • Table 4 Analysis of GP+envAml2 clones harbouring a single copy of a recombinant CD2-LCR comprising retrovirus construct, obtained through infection with ecotropic recombinant retrovirus supernatant.
  • a 5' element of the chicken b-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74, 505-514.
  • Retrovirus-mediated gene transfer into embryonal carcinoma and haemopoietic stem cells expression from a hybrid long terminal repeat. Gene 84, 419- 427.
  • Van Beusechem V.W., Kukler, A., Einerhand, M.P.W., Bakx, T.A., van der Eb, A.J. , van Bekkum, D.W. and Valerio, D. (1990) .
  • Van Beusechem v.w., Kukler, A., Heidt, P.J. and Valerio, D. (1992). Long-term expression of human adenosine deaminase in rhesus monkeys transplanted with retrovirus-infected bone- marrow cells. Proc. Natl. Acad. Sci. USA 89, 7640-7644.
  • Van Beusechem V.W., Bakx, T.A., Bart-Baumeister, J.A.K. , Braakman, E. , Kaptein, L.C.M., Kukler, A. and Valerio, D. Retrovirus-mediated gene transfer into rhesus monkey haemopoietic stem cells: the effect of viral titers on transduction efficiency. Hum.Gene Ther.4 (1993) 239-247.
  • Van Beusechem V.W. , Bart-Baumeister, J.A.K. , Hoogerbrugge, P.M. and Valerio, D. Influence of Interleukin-3, Interleukin-6 and Stem Cell Factor on retroviral transduction of rhesus monkey CD34+ haematopoietic progenitor cells measured in vitro and in vivo. , Gene Ther.2 (1995) 1-11.
  • Herpes simplex virus thymidine kinase gene therapy for rat malignant brain tumors Hum.Gene Ther. 7, 197-205.
  • Figure 1 Schematic representation of the cloning strategy used to construct pLec. LTR, long terminal repeat;HIII, Hindlll,-mcs, multiple cloning site; SD, splice donor site; SA, splice acceptor site; TAG, stop codon; waved lines represent mouse genomic flanking sequences.
  • Figure 2 Schematic representation of the cloning strategy used to construct pLec ⁇ Mo. x, Xbal and see legend figure 1.
  • FIG. 1 Schematic representation of the cloning strategy used to construct pLTKkoz. see also legend figure 1.
  • Figure 4a and b Schematic representation of the constructs pIGTK and pLTT ⁇ Mo. see also legend to figure 1.
  • IL2 producer cells (— - - — ) .
  • One group was injected with IG-RV-TK producer cells without subsequent GCV treatment (- - - - - -) .
  • Five days after virus injection 15 mg/kg GCV was administered twice a day for ten days i.p..
  • IG-RV-TK treated rats lived significantly longer than controls (p ⁇ 0.01; log rank test) .
  • One rat died of superficial leptomeningeal tumour (*). This rat is censored in the survival analysis.
  • Figure 6 Schematic representation of the constructs used to test the intercistronic linkers. TGA, stop codon; E, EcoRI; S, Sail.
  • Figure 6A Bar diagram representing the average number of colonies obtained after G418 selection of transfected Rat-2 cells corrected for (arbitrary units of) Luciferase activity.
  • Figure 6B Bar diagram representing the average number of colonies obtained after G418 selection of transfected Rat-2 cells corrected for the number of colonies obtained after selection in HAT medium.
  • FIG. 7 Rh-123 exclusion analysis on A2780 cells after transduction with IGmdrl-G virus supernatant. Transduced A2780 cells (A) are compared to mock-infected controls (B) .
  • FIG. 8 Vincristine survival of CD34 + peripheral blood cells after transduction with IGmdrl-G virus supernatant.
  • CD34 selected PBPC were transduced with (squares) or without (circles) IGmdrl-G supernatant for 4 days in the presence of IL-3 and 4 ⁇ g/ml protamine sulphate. Supernatant was refreshed daily.
  • PBSC were seeded for in vitro colony formation (GM-CFU) in the presence of increasing amounts of vincristine. Colonies were scored after 14 days. The survival was calculated by dividing the number of GM-CFU in the dishes with drugs by the number of GM-CFU in the dishes without drugs.
  • Figure 9 Physical map of retroviral construct IG-GC-1.
  • SD splice donor: SA, splice acceptor.
  • Figure 10 Physical map of retroviral construct IG-GC-2. SD, splice donor; SA, splice acceptor; TAG; mutated startcodon of gag coding sequence; LTR, long terminal repeat.
  • Figure 11 PCR strategy to delete part of the 3'- untranslated region of the human Glueocerebrosidase cDNA (see text for details) .
  • Figure 12. Physical map of retroviral construct IG-GC-3. SD, splice donor; SA, splice acceptor; TAG, mutated startcodon of gag coding sequencel; LTR, long terminal repeat.
  • Figure 13 Physical map of retroviral construct IG-GC-4. SD, splice donor; SA, splice acceptor; TAG, mutated startcodon of gag coding sequence; LTR, long terminal repeat.
  • Figure 14A Increase in glueocerebrosidase enzyme activity after transfection of retroviral constructs IG-GC-2 and IG-GC- 4 in packaging cell lines GP + E 86 and Psi-CRE respectively and after infection of 3T3 cells.
  • FIG. 14B Western blot with primate specific monoclonal antibody 8E4 (80 mg total protein/lane).
  • A, B, C Cell lysates prepared from GP + E86 cells after transfection with retroviral construct IG-GC-2. +/- : With or without protease inhibitors.
  • GP Cell lysate prepared from non-transfected GP + E86 cells.
  • FIG. 15 Western blot with primate specific monoclonal antibody 8E4 (80 mg total protein/lane).
  • B1/B2 Cell lysates prepared from PA317 cells after repeated infection (1, 4, 10 times) with ecotropic GP2b virus (duplicate) .
  • GP cell lysate prepared from GP - E86 cells after transfection with retroviral construct IG-GC-2 as positive control.
  • PA Cell lysate prepared from non-infected PA317 cells. Cer: 16.5 mg (0.7 units) recombinant glueocerebrosidase (Genzyme Corp.) as positive control.
  • FIG. 16 Western analysis on hGC expression in producer (PA2, PA4, PG2 and PG4) and of the parental cell line- PA317 (PA) was loaded on a 10% acrylamide gel. As positive control approx. 1 unit of Cerezyme (Genzyme corp.) was loaded. B) 20 ⁇ g total protein of each infected Gaucher type II cell pool (PA2, PA4, PG2 and PG4) and of the parental cell line (T-II) was loaded.
  • Figure 17. DNA analysis of PA2 infected individual 3T3 cell clones . Isolated DNA was digested with EcoRI (unique restriction site just 5' of hGC sequence) and Southern blots were probed with the complete hGC sequence. In the genomic DNA of each individual 3T3 clone a mean of three bands is visible resulting in an estimated functional titer of at least 3 x 10 5 /ml.
  • Figure 18 hGC-activity assay (PNP- ⁇ -Glu) on normal and Gaucher type I and II fibroblasts as well as on type I and II Gaucher fibroblasts infected with virus supernatant.
  • FIG. 20 PCR and subsequent Southern analysis show the presence of the recombinant provirus in infected CD34 4" Gaucher bone marrow cells. Southern blot was probed with a 300 bp BamHI fragment from the 3' end of the hGC gene.
  • Figure 21 Strategy for the introduction of a point mutation (Phe32Ser) in the human dihydrofolate reductase cDNA.
  • Figure 22 Schematic drawing of the map of recombinant retroviral vectors IG-GC5 and IG-GC6.

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Abstract

La présente invention se rapporte au domaine de la biologie moléculaire, en particulier à la technologie de l'ADN recombiné, notamment aux vecteurs rétroviraux. Les vecteurs rétroviraux constituent des véhicules appropriés pour transférer un matériel génétique à étudier dans certaines cellules selon des 'stratégies de thérapie génique'. Cependant, les vecteurs rétroviraux décrits jusqu'à présent ne sont pas idéaux. Ils peuvent donner lieu à des phénomènes de recombinaison qui donnent des virus assistants (pathogènes), et peuvent exprimer des matériels génétiques protéiniques viraux conduisant à des réponses immunitaires, etc. Ces inconvénients, entre autres, peuvent être supprimés au moyen des vecteurs, cellules, nécessaires et procédés de la présente invention, par la création d'un vecteur dérivé d'un rétrovirus, comprenant une séquence responsable de la commande transcriptionnelle, y compris un amplificateur. Ce vecteur comprend en outre un site d'insertion d'au moins un gène à étudier, un signal d'encapsidation, ledit vecteur n'ayant aucune séquence rétrovirale superflue et aucun cadre de lecture ouvert codant au moins des parties des protéines virales, et ledit vecteur étant caractérisé en ce que l'amplificateur est actif dans des cellules indifférenciées.
EP96915215A 1995-05-10 1996-05-07 Ameliorations apportees a des vecteurs retroviraux, appropries en particulier pour la therapie genique Withdrawn EP0835320A1 (fr)

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EP95201211 1995-05-10
EP95201211 1995-05-10
EP96915215A EP0835320A1 (fr) 1995-05-10 1996-05-07 Ameliorations apportees a des vecteurs retroviraux, appropries en particulier pour la therapie genique
PCT/NL1996/000195 WO1996035798A1 (fr) 1995-05-10 1996-05-07 Ameliorations apportees a des vecteurs retroviraux, appropries en particulier pour la therapie genique

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JP (1) JPH11511651A (fr)
AU (1) AU5703796A (fr)
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WO (1) WO1996035798A1 (fr)

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CA2288328A1 (fr) 1997-05-13 1998-11-19 University Of North Carolina At Chapel Hill Vecteurs de transfert de gene comportant un lentivirus
US20030017138A1 (en) 1998-07-08 2003-01-23 Menzo Havenga Chimeric adenoviruses
US6492169B1 (en) 1999-05-18 2002-12-10 Crucell Holland, B.V. Complementing cell lines
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
WO2001002551A2 (fr) * 1999-06-30 2001-01-11 Evotec Oai Ag Particules de type viral, preparation et utilisation de ces dernieres, de preference dans le criblage pharmaceutique et la genomique fonctionnelle
US20040014033A1 (en) 1999-06-30 2004-01-22 Evotec Biosystems Ag, A Corporation Of Germany Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
US7476517B2 (en) 1999-06-30 2009-01-13 Evotec Ag Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
CN1980955A (zh) * 2004-04-29 2007-06-13 北卡罗来纳-查佩尔山大学 增强细胞粘连特性的方法和组合物
WO2008108344A1 (fr) * 2007-03-02 2008-09-12 Cellgentech, Inc. Cellule pour thérapie génique destinée à traiter la déficience en lcat, et vecteur rétroviral non réplicatif et plasmide servant à produire ladite cellule

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FR2699191B1 (fr) * 1992-12-16 1995-02-10 Univ Paris Curie Nouveaux vecteurs rétroviraux, lignées cellulaires les contenant, et leur utilisation dans le traitement des tumeurs.
GB9305759D0 (en) * 1993-03-19 1993-05-05 Medical Research Council And T Hiv therapy method and agents
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US5631162A (en) * 1993-06-11 1997-05-20 Massachusetts Institute Of Technology Retroviral vectors for transducing β-globin gene and β-locus control region derivatives
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WO1996035798A1 (fr) 1996-11-14
JPH11511651A (ja) 1999-10-12
CA2218808A1 (fr) 1996-11-14
AU5703796A (en) 1996-11-29

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