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WO1994029437A1 - Vecteurs retroviraux exempts de region u3, exempts de recombinaison, a auto-inactivation, hautement efficaces - Google Patents

Vecteurs retroviraux exempts de region u3, exempts de recombinaison, a auto-inactivation, hautement efficaces Download PDF

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Publication number
WO1994029437A1
WO1994029437A1 PCT/US1994/006415 US9406415W WO9429437A1 WO 1994029437 A1 WO1994029437 A1 WO 1994029437A1 US 9406415 W US9406415 W US 9406415W WO 9429437 A1 WO9429437 A1 WO 9429437A1
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host cell
ltr
promoter
retroviral
vector
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PCT/US1994/006415
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English (en)
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Ralph C. Dornburg
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University Of Medicine & Dentistry Of New Jersey
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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
    • 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

Definitions

  • This invention relates to recombination-free, highly-efficient retroviral vectors that self-inactivate after one round of retroviral replication.
  • the present vectors allow cell-type specific gene expression from tissue specific promoters and enhancers.
  • the recombination-free retroviral vectors eliminate risks of downstream activation of cellular proto-oncogenes (tumorigenesis) that can occur with conventional vectors.
  • This invention also relates to a retrovirus produced by the recombinant retrovirus vector and a host cell containing the provirus of the present invention.
  • Retroviral vectors are the most efficient tools to introduce genes into vertebrate cells. Clinical experiments have been conducted to use retrovirus vectors to cure a genetic disease in humans (adenosine deaminase (ADA) deficiency). Besides correcting inborn errors of metabolism, gene therapy is also being tested in clinical trials to cure cancer and various other diseases (Science 1992, Vol. 258, pp. 744-746).
  • ADA adenosine deaminase
  • Retroviral vectors are basically retroviral particles that contain a crippled viral genome in which all viral protein coding sequences have been replaced with the gene(s) of interest. As a result, such viruses cannot further replicate after one round of infection without the help of a helper virus. Retroviral vector particles are produced by helper cells ( Figure 1). Such helper cells contain plasmid constructs which express all retroviral proteins necessary for particle production and replication. After the introduction (transfection) of the retroviral vector genome into such helper cells, the vector genome (an RNA genome) is encapsidated into virus particles (due the presence of specific encapsidation sequences). Virus particles are released from the helper cell carrying a genome containing only the gene(s) of interest ( Figure 1).
  • RNA genome After infection of a fresh target cell, the RNA genome is reverse transcribed into DNA.
  • the DNA copy is integrated into the host genome ( Figure 2A).
  • the integrated viral DNA is called the provirus.
  • retroviral vector systems derived from chicken or murine retroviruses, have been developed for the expression of various genes (for reviews see Temin, 1987; Gilboa, 1990).
  • Retroviral vectors have several limitations. For example, one major concern is the possible activation of proto-oncogenes as a result of the integration of the vector into the target cell genome. The activation of proto-oncogenes which is mediated by the viral LTR (Long Terminal Repeats) promoter and enhancer can lead to the malignant transformation (cancer) of the infected cell. Another problem with current retroviral vectors is regulated gene expression. Due to the presence of the retroviral LTR promoter and enhancer, it is impossible to design reliable vectors for tissue specific gene expression.
  • LTR Long Terminal Repeats
  • retroviral vectors have been developed from murine leukemia virus (MLV) and spleen necrosis virus (SNN) that self-inactivate after one round of retroviral replication (Yu et al, 1986; Dougherty and Temin, 1987; United States patent no. 4,980,289 to Temin et al.). This has been achieved by deleting parts or almost all sequences of the retroviral U3 region of the right LTR ( Figure 2B). As a result of the mechanism of the retroviral life- cycle, after one round of replication, a retroviral provirus is formed that does not contain LTR promoters.
  • MMV murine leukemia virus
  • SNN spleen necrosis virus
  • DS ⁇ cells contain two separate plasmids for the expression of gag-pol and env proteins (Dougherty and Temin, 1989).
  • One plasmid contains the gag-pol gene of S ⁇ V which is expressed from the CMV promoter.
  • the other plasmid contains the envelope gene unit of S ⁇ N which is expressed from the RSV promoter.
  • FIGURE 1 is a diagram illustrating retroviral helper cells producing recombination-free, self-inactivating retroviral vectors.
  • FIGURE 2 is a diagram illustrating retroviral vector replication.
  • FIGURE 3 is a diagram illustrating retroviral vectors derived from spleen necrosis virus (SNV).
  • FIGURE 4 is a diagram illustrating the nucleotide sequences at the junction of the cytomegalovirus (CMV) with spleen necrosis virus sequences as present in the pPOll l and pPO115 vector series.
  • CMV cytomegalovirus
  • the present invention pertains to a recombinant retrovirus vector having a U3-free 5' LTR, a partially deleted 3' LTR, all essential cis-acting sequences for replication, an internal promoter recognizable by a selected host cell, and a non-retroviral gene under the control of the recognized promoter wherein:
  • the U3-free 5 * LTR is positioned 5' of the non-retroviral gene and has a transcriptional promoter and enhancer different from that of the original retroviral promoter and enhancer replacing the original U3 region of the 5 'LTR;
  • the partially deleted 3' LTR is positioned 3' of the non-retroviral gene and has no U3 sequences except for those required at the attachment site for viral integration;
  • the internal recognized promoter is positioned adjacent to the non-retroviral gene on the vector to permit expression of the non-retroviral gene in the host cell; whereby the vector can produce progeny virus in a helper cell with the progeny virus being capable of infecting the selected host cell and forming a provirus in the host cell, with the non-retroviral gene being expressible in the host cell, but the provirus in the host cell will be replication incompetent even in the presence of a helper virus.
  • the present invention also pertains to a retrovirus produced by the recombinant retrovirus vector and a host cell containing the provirus of the present invention.
  • This invention relates to recombination-free, highly-efficient retroviral vectors that self-inactivate after one round of retroviral replication.
  • the vectors allow cell-type specific gene expression from tissue specific promoters and enhancers and eliminate risks of downstream activation of cellular proto-oncogenes (tumorigenesis) that can occur with conventional vectors.
  • the present self- inactivating vectors eliminate the problems of recombination and give rise to high gene transfer efficiency, up to 1,000 fold higher than that of existing self- inactivating vectors.
  • the vectors described herein are derived from spleen necrosis virus but vector modification may be made with any retroviral vector leading to similar results.
  • the novel vectors have a high potential for use in human gene therapy and in other gene transfer applications in which the cell-type specific gene expression is required, e.g., tissue specific gene expression in transgenic animals.
  • the U3 region of the left LTR was replaced by the immediate early gene promoter and enhancer of the cytomegalovirus (CMV, Figures 3 and 4). This promoter was shown to be the strongest among a series of promoters tested for gene expression in a large variety of cell-types including the SNN promoter in D17 cells.
  • CMV cytomegalovirus
  • the substitution of the U3 promoter by the CMV promoter was done in such a way that initiation of transcription started at the beginning of R as in the wild-type virus.
  • the vector is a recombinant retrovirus vector having a normally replication incompetent retrovirus gene sequence with a foreign eukaryotic gene.
  • the retrovirus gene sequence is prepared with a deficiency in the retrovirus promoter so that the vector can still produce progeny virus in a helper cell, with the progeny virus being capable of infecting a selected eukaryotic host cell, forming a provirus, and expressing the eukaryotic gene in the host cell, but the provirus will be defective in the retrovirus promoter sequence.
  • the vector is a recombinant plasmid.
  • a foreign internal promoter is positioned adjacent to the foreign eukaryotic gene on the vector to permit expression of the foreign eukaryotic gene in the eukaryotic host cell without initiating retroviral provirus gene expression.
  • the reading direction of the foreign promoter is inverted relative to the normal reading direction of the retrovirus gene sequence, and a foreign 3' RNA processing sequence is positioned on the side of the foreign eukaryotic gene sequence which is opposite to the foreign promoter.
  • the retrovirus is a normally replication incompetent retrovirus of the type having a retrovirus portion and a foreign eukaryotic portion, the retrovirus portion having a deficient promoter portion, such that the virus is capable of infecting a eukaryotic host cell, forming a provirus, and expressing a eukaryotic protein coded for by the foreign eukaryotic portion in the host cell, but the provirus will be defective in a retrovirus promoter such that retroviral provirus gene expression doesn't take place in the host cell.
  • the present invention allows one to select a eukaryotic gene of interest, insert the gene into a vector designed in accordance with the present invention, transfect a helper cell with the vector, harvest virus stock from the helper cell, use the harvested progeny virus to infect a target cell, and have the proviruses which are formed in the target cells express the inserted eukaryotic gene without expressing any retroviral proteins. Since there is no retroviral promoter that is active on the provirus, endogenous helper proteins cannot trigger production of a virus from the provirus. Since there is no retroviral promoter in the provirus, the provirus cannot provide a retrovirus signal that might trigger the host cell to act in an unintended way. The lack of retroviral promoter stops production of retroviral RNA. This system renders much more likely the acceptability of recombinant retrovirus as drugs for vertebrates.
  • the enhancer and the promoter sequences of the retrovirus present at U3 in SNV have been deleted from the right side of the DNA sequence in the plasmid vector (3 'LTR). Only the sequences required at the attachment site for viral integration in the 3' LTR are present.
  • the U3 5' LTR has a transcriptional promoter and enhancer different from that of the original retroviral promoter and enhancer to replace the original U3 region of the 5 'LTR.
  • the total lack of homology in the two U3 sequences prevent recombination.
  • the U3 sequence normally present in the 5' LTR has been reported to be used as a template to repair a partially deleleted U3 sequence in the 3' LTR.
  • the vector DNA is used to transfect helper cells in a conventional manner. Because transcription from the vector begins at R on the left side, and because the romoter on the left side is not defective, virus can be harvested from the transfected helper cell in the conventional manner. Target cells can then be infected with the harvested virus. Since the right side U3 supplies the coding sequences for both U3 segments in the resulting provirus, the transcriptional promoter which was originally deleted on one side of the plasmid DNA shows up as being deleted from both sides in the resulting provirus. The vector therefore permits a stock of the retrovirus progeny virus to be grown up, yet will not permit further replication after one infection cycle.
  • an internal promoter is not inserted, there will be no promoter to produce the desired foreign eukaryotic gene expression.
  • a promoter is positioned immediately adjacent to the foreign eukaryotic of interest so that no intervening retrovirus genes RNA is expressed. Because deletion of most of the U3 in SNV resulted in a loss of correct 3' end processing of viral RNA, even though AAUAAA was still present, a polyadenylation site was added to the vector. Moreover, problem recombinations are unlikely because the U3 sequences in the vector are not homologous.
  • Abbreviations used in the present invention are as follows: pro- promoter; enh-enhancer; PBS-primer binding site for DNA synthesis; PPT- polypurine track for DNA synthesis; E-encapsidation sequences for RNA packaging; attR+-a sequence that will form the right side of the attachment site relating to integration; attL+-the sequence that will form the left side of the attachment side relating to integration; attL-f-the deletion of the original provirus left-side attachment site; and attR+-the deletion of the original right side attachment site.
  • oligonucleotide refers to primers, probes, oligomer fragments to be detected, oligomer controls, and unlabeled blocking oligomers. Oligonucleotide are molecules comprised of two or more deoxyribonucleotides or ribonucleotides.
  • primer refers to an oligonucleotide, preferably an oligodeoxyribonucleotide, either naturally occurring such as a purified restriction digest or synthetically produced, which is capable of acting as a point of initiation of synthesis when subjected to conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e. , in the presence of nucleotides, an agent for polymerization such as a DNA polymerase, and a suitable temperature and pH.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent.
  • FIGURE 1 is a diagram illustrating retroviral helper cells producing recombination-free, self-inactivating retroviral vectors.
  • FIGURE IB shows that after the transfection of the retroviral vector genome into such helper cells, viral particles are produced that contain the RNA vector genome with the gene of interest.
  • FIGURE 2 is a diagram illustrating retroviral vector replication.
  • FIGURE 2A shows an example of a conventional retroviral vector provirus as present in the helper cell shown at the top.
  • This vector contains wild-type long terminal repeats with full-length U3, R, and U5 regions.
  • RNA transcription yields the RNA transcript shown below.
  • E encapsidation sequence
  • ppt polypurine tract
  • FIGURE 2B illustrates the principle of a self-inactivating retroviral vector.
  • the vector present in the helper cell is shown at the to]). This vector is almost identical to the one shown above. However, the long terminal repeat at the right side has been modified in the following way: most of U3-sequences including the promoter and enhancer sequences have been deleted. Maintenance of some U3-sequences that form the attachment site (att) is essential for the efficient integration of the vector genome.
  • the RNA transcribed from the U3-minus vector virus does not contain the retroviral promoter and enhancer. Thus, after one round of retroviral replication, a promoter-less retroviral provirus is formed. Genes can be expressed from internal tissue-specific promoters (pro).
  • FIGURE 3 is a diagram illustrating retroviral vectors derived from spleen necrosis virus (SNV).
  • SNV spleen necrosis virus
  • the SNV genome provirus
  • pJD214HY An example of a standard retroviral derived from SNV is shown below (pJD214HY).
  • pJD214HY An example of a standard retroviral derived from SNV is shown below (pJD214HY).
  • the protein coding regions gag-pol and env, the region from Sail to the 3' end of env
  • hygro hygromycin resistance gene
  • pJD220SVHY is a first generation self-inactivating retroviral vector in which the retroviral promoter and enhancer of the right LTR have been deleted (designated as U3 minus; for more details, see Figure 2B).
  • U3 minus the retroviral promoter and enhancer of the right LTR have been deleted
  • Figure 2B the polyadenylation sequence of simian virus 40 (SV40ter) has been inserted downstream of the U3-minus LTR.
  • a universal, second generation U3-free vector contains a multiple cloning site replacing the promoter (SV40pro) and hygromycin B resistance gene (hygro) as present in pJD220SVHY and pPOl l l-Rl (from Eagl to Clal).
  • the multiple cloning site has been derived from pBluescript II (Eagl to Clal) and allows the easy insertion of various genes and promoters to give new retroviral vectors.
  • FIGURE 4 is a diagram illustrating the nucleotide sequences at the junction of the cytomegalovirus (CMV) with spleen necrosis virus sequences as present in the pPOlll and pPO115 vector series. An except of the S ⁇ V genome showing the TATA box and initiation of transcription is shown at the top. The Sacl site downstream of the TATA box of the CMV immediate early promoter (shaded sequence) was used for cloning to connect the CMV promoter and enhancer to the SNN sequences.
  • pPOl ll-R2 and ⁇ POl l l-R3 are 3 or 9 nucleotides shorter than pPOl ll-Rl, respectively.
  • the function of the new vectors was tested in a tissue culture system as follows and as described earlier (Olson et al., 1992): The new vector constructs pPOlll-Rl, pPOlll-R2, and pPOlll-R3 were transfected into helper cells.
  • the retroviral vectors pJD214HY a standard retroviral vector with two complete wild-type LTRs, and pJD220SVHY, the first generation self-inactivating vector from which the new vectors have been derived (Figure 3)
  • Virus was harvested from confluent step 1 cultures and fresh helper cells were infected. The infected helper cells were termed step 2 cells. Virus titers were determined (Table 1). We show that the new vectors pPOlll-Rl to pPOlll-R3 were more efficient than pJD220SVHY. They were almost as efficient as a vector with two wild-type LTRs (pJD214HY).
  • virus particles were harvested from confluent step 2 cell cultures (mass infection) and fresh D17 cells were infected. Detection of hygromycin resistant colonies indicates that recombination reconstituted the U3 region: due to the lack of control sequences in R ⁇ A transcripts derived from completely U3-minus proviruses, such vectors are not further passaged by retroviral proteins. Thus, only vectors with complete, repaired LTRs are transferred to new target cells.
  • pJD220SVHY was further passaged with high efficiency confirming earlier findings (Olson et al., 1992).
  • VECTOR NAME 1.INFECTION 2. INFECTION pJD214HY 6 X 105 5 X 106
  • the retroviral vectors were transfected into helper cells followed by hygromycin selection.
  • Virus was harvested from confluent cultures and fresh helper cells were infected (referred to as first infection). Infected cells were selected for hygromycin resistance, and virus titers were determined (expressed as colony forming units (CFU) per ml of supernatant medium). Cell- lines were established from infected tissue culture plates which contained more than 1,000 independent hygromycin resistant colonies.
  • Virus was harvested from confluent cell cultures and fresh D17 cells were infected (referred to as second infection). Infected cells were selected for hygromycin resistance and virus titers were determined.
  • CMV immediate early promoter and enhancer of cytomegalovirus
  • Plasmid pPO102 was digested with Sacl and re-ligated after treatment with Klenow polymerase 1 (creation of blunt ends) to eliminate a Sacl site present in the encapsidation sequence. The resulting plasmid was termed (pPO102-S).
  • a DNA fragment termed Rl comprises map units 383 to 986 of the SNV genome
  • fragment R2 comprises map units 385 to 986
  • fragment R3 comprises map units 392 to 986.
  • All PCR primers contained Sacl recognition sites at their 5' ends. After Sacl digestion, fragments Rl, R2, and R3 were cloned into the Sacl site of pBluescript II KS. The resulting plasmids were termed pPO103-Rl, pPO103-R2 and pPO103-R3, respectively.
  • Fragments Rl to R3 were isolated from such plasmids and cloned into the Sacl site of pPOlOl.
  • the resulting plasmids were termed pPO104-Rl, pPO104-R2, and pPO104-R3, respectively.
  • Plasmid pJD220SVHY (Dougherty and Temin, 19986) was digested with Sspl and Sacl. After Klenow poll treatment, a linker coding for the recognition site of the restriction enzyme Bgl2 was ligated. This procedure removed the left LTR promoter, and all of the encapsidation sequences present in this vector. The resulting plasmid was termed pPO106.
  • DNA fragments derived from plasmids pPO104-Rl, pPO104-R2, and pPO104-R3 comprising the CMV promoter, R, U5, and the encapsidation sequence (Bgl2 fragments) were isolated and inserted into plasmid pPO106 digested with Bgl2.
  • the resulting plasmids were termed pPOlll-Rl, ⁇ POl l l-R2, and pPOl ll-R3, respectively.
  • the universal retrovirus vectors pPO115-Rl and PPO1 15-R2 were made by replacing the SV40 promoter and hygromycin B phosphotransferase gene of pPOlll-Rl and pPOlll-
  • a promoter-less retroviral vector indicates that there are sequences in U3 required for 3 'RNA processing. Proc. Natl. Acad. Sci. USA 84, 1197-1201.
  • Embretson,J.E., and H.M. Temin. 1987 Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis encapsidation-minus RNAs by the reticulo-endotheliosis virus helper cell line. J.Virol. 61,2675-2683.

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Abstract

La présente invention se rapporte à des vecteurs de rétrovirus de recombinaison possédant une répétition terminale longue (RTL) 5' exempte de région U3, une répétition terminale longue (RTL) 3' partiellement modifiée, toutes les séquences essentielles de cis-activation destinées à la réplication, un promoteur interne reconnaissable par une cellule hôte sélectionnée, et un gène non rétroviral se trouvant sous la régulation du promoteur reconnu, vecteurs dans lesquels (a) la répétition terminale longue (RTL) 5' exempte de région U3 se trouve à la position 5' du gène non rétroviral et possède un promoteur et un activateur transcriptionnels, différents du promoteur et de l'activateur rétroviraux, d'origine, remplaçant la région U3 d'origine de la répétition terminale longue (RTL) 5'; (b) la répétition terminale longue (RTL) 3' partiellement modifiée se trouve à la position 3' du gène non rétroviral et ne possède pas de séquences U3, excepté pour celles nécessaires sur le site de fixation concernant l'intégration virale; (c) une séquence de signal d'addition de polyadénylation exogène reconnue par la cellule hôte sélectionnée, et positionnée sur le vecteur 3' par rapport au site d'intégration virale de la répétition terminale longue (RTL) 3'; et (d) le promoteur interne reconnu est positionné à côté du gène non rétroviral sur le vecteur afin de permettre l'expression du gène non rétroviral dans la cellule hôte. Le vecteur peut produire un virus de la descendance dans une cellule auxiliaire, le virus de la descendance étant capable d'infecter la cellule hôte sélectionnée et de former un provirus dans la cellule hôte, le gène non rétroviral pouvant être exprimé dans la cellule hôte, mais le provirus de la cellule hôte sera inapte à la réplication, même en présence d'un virus assistant.
PCT/US1994/006415 1993-06-07 1994-06-07 Vecteurs retroviraux exempts de region u3, exempts de recombinaison, a auto-inactivation, hautement efficaces WO1994029437A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021912A1 (fr) * 1994-02-14 1995-08-17 The Macfarlane Burnet Centre For Medical Research Limited Souches non pathogenes de vih-1
WO1996035798A1 (fr) * 1995-05-10 1996-11-14 Introgene B.V. Ameliorations apportees a des vecteurs retroviraux, appropries en particulier pour la therapie genique
WO1996037623A1 (fr) * 1995-05-22 1996-11-28 Oxford Biomedica (Uk) Limited Vecteurs retroviraux
WO1997013867A1 (fr) * 1995-10-13 1997-04-17 Bavarian Nordic Research Institute Vecteurs retroviraux porteurs d'inhibiteurs 1 derives de cellules senescentes (sdi-1) ou de sequences de nucleotides de sdi-1 antisens
US6015661A (en) * 1994-02-14 2000-01-18 The Macfarlane Burnet Centre For Medical Research Limited Methods for the detection of non-pathogenic HIV-1 strains containing deletions in the Nef coding region and U3 region of the LTR
US6027721A (en) * 1996-05-20 2000-02-22 Cytotherapeutics, Inc. Device and method for encapsulated gene therapy
US6540995B1 (en) 1996-03-27 2003-04-01 Bavarian Nordic Research Institute Gmbh Encapsulated cells producing cytochrome P450
CN1117156C (zh) * 1994-09-02 2003-08-06 Gsf环境与健康研究中心有限公司 非自动失活的定位表达的逆转录病毒载体
US6776985B1 (en) 1995-06-27 2004-08-17 Bavarian Nordic A/S Encapsulated cells producing viral particles
US6783977B1 (en) * 1997-04-28 2004-08-31 Institut National De La Sante Et De La Recherche Medicale (Inserm) Internal ribosome entry site and vector containing same
US6893634B1 (en) 1996-03-27 2005-05-17 Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Encapsulated cells producing cytochrome P450
US7022319B1 (en) 1995-03-09 2006-04-04 Gsf - Forschungszentrum Fuer Umwelt Und Gesundheit Gmbh Vectors carrying therapeutic genes encoding antimicrobial peptides for gene therapy
US7074398B1 (en) 1995-10-13 2006-07-11 Gsf-Forschungszentrum Fuer Umwelt Und Gesundheit Gmbh Retroviral vectors carrying senescent cell derived inhibitors 1 (SDI-1)or antisense SDI-1 nucleotide sequences

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124263A (en) * 1989-01-12 1992-06-23 Wisconsin Alumni Research Foundation Recombination resistant retroviral helper cell and products produced thereby

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124263A (en) * 1989-01-12 1992-06-23 Wisconsin Alumni Research Foundation Recombination resistant retroviral helper cell and products produced thereby

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF VIROLOGY, Volume 66, Number 3, issued March 1992, OLSON et al., "Unusually High Frequency of Reconstitution of Long Terminal Repeats in U3-Minus Retrovirus Vectors by DNA Recombination or Gene Conversion", pages 1336-1343. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6015661A (en) * 1994-02-14 2000-01-18 The Macfarlane Burnet Centre For Medical Research Limited Methods for the detection of non-pathogenic HIV-1 strains containing deletions in the Nef coding region and U3 region of the LTR
WO1995021912A1 (fr) * 1994-02-14 1995-08-17 The Macfarlane Burnet Centre For Medical Research Limited Souches non pathogenes de vih-1
CN1117156C (zh) * 1994-09-02 2003-08-06 Gsf环境与健康研究中心有限公司 非自动失活的定位表达的逆转录病毒载体
US7531353B1 (en) 1994-09-02 2009-05-12 Gsf-Forschungszentrum Fur Unwelt Und Gesundheit Gmbh Non self-inactivating, expression targeted retroviral vectors
US7022319B1 (en) 1995-03-09 2006-04-04 Gsf - Forschungszentrum Fuer Umwelt Und Gesundheit Gmbh Vectors carrying therapeutic genes encoding antimicrobial peptides for gene therapy
WO1996035798A1 (fr) * 1995-05-10 1996-11-14 Introgene B.V. Ameliorations apportees a des vecteurs retroviraux, appropries en particulier pour la therapie genique
WO1996037623A1 (fr) * 1995-05-22 1996-11-28 Oxford Biomedica (Uk) Limited Vecteurs retroviraux
EP1164196A1 (fr) * 1995-05-22 2001-12-19 Oxford Biomedica (UK) Limited Vecteurs rétroviraux
US6776985B1 (en) 1995-06-27 2004-08-17 Bavarian Nordic A/S Encapsulated cells producing viral particles
US7074398B1 (en) 1995-10-13 2006-07-11 Gsf-Forschungszentrum Fuer Umwelt Und Gesundheit Gmbh Retroviral vectors carrying senescent cell derived inhibitors 1 (SDI-1)or antisense SDI-1 nucleotide sequences
WO1997013867A1 (fr) * 1995-10-13 1997-04-17 Bavarian Nordic Research Institute Vecteurs retroviraux porteurs d'inhibiteurs 1 derives de cellules senescentes (sdi-1) ou de sequences de nucleotides de sdi-1 antisens
US6540995B1 (en) 1996-03-27 2003-04-01 Bavarian Nordic Research Institute Gmbh Encapsulated cells producing cytochrome P450
US6893634B1 (en) 1996-03-27 2005-05-17 Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Encapsulated cells producing cytochrome P450
US6027721A (en) * 1996-05-20 2000-02-22 Cytotherapeutics, Inc. Device and method for encapsulated gene therapy
US6783977B1 (en) * 1997-04-28 2004-08-31 Institut National De La Sante Et De La Recherche Medicale (Inserm) Internal ribosome entry site and vector containing same

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