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WO2000046360A1 - Amelioration du systeme vecteur dependant d'un assistant-dependant pour la therapie genique - Google Patents

Amelioration du systeme vecteur dependant d'un assistant-dependant pour la therapie genique Download PDF

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Publication number
WO2000046360A1
WO2000046360A1 PCT/US2000/002405 US0002405W WO0046360A1 WO 2000046360 A1 WO2000046360 A1 WO 2000046360A1 US 0002405 W US0002405 W US 0002405W WO 0046360 A1 WO0046360 A1 WO 0046360A1
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helper
packaging signal
virus
adenovirus
dependent
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PCT/US2000/002405
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English (en)
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Andrew Bett
Volker Sandig
Rima Youil
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Merck & Co., Inc.
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Priority to CA002360796A priority Critical patent/CA2360796A1/fr
Priority to US09/890,836 priority patent/US7132277B1/en
Priority to JP2000597420A priority patent/JP2002535986A/ja
Priority to EP00910029A priority patent/EP1151091A4/fr
Publication of WO2000046360A1 publication Critical patent/WO2000046360A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10344—Chimeric viral vector comprising heterologous viral elements for production of another viral vector

Definitions

  • This invention relates to adenoviral vectors which are useful for nucleic acid delivery into a cell such as in gene delivery and nucleotide vaccine applications. It also relates to host cells and methods using these vectors.
  • Adenoviral vectors provide a vehicle for introducing nucleic acids into a cell in vitro and in vivo. Over 100 different serotypes, of which 47 are of human origin, have been reported. (Hierholzer, et al., J. infect. Dis. 759:804-813, 1988.) Adenoviruses type 2 and 5 used to produce adenoviral vectors are well characterized.
  • a human adenovirus contains a 5' inverted terminal repeat (ITR); a packaging signal; region El made up of the early regions El A and E1B; region E2; region E3, region E4; the late regions L1-L5; and a 3' ITR.
  • Regions El and E4 contain regulatory proteins, region E2 encodes proteins required for replication, and the L region encodes for the structural proteins of the virus.
  • the E3 region is dispensable for virus growth in vitro. (Hitt, et al, Advances in Pharmacology 40: 137-206, 1996.)
  • a replicating viral vector based on the adenovirus must contain adenovirus cis elements needed for replication.
  • Packaging also requires cis elements on the vector whereas the necessary proteins can be supplied in trans. In trans supplementation can be brought about by specialized cells and/or additional viruses producing the needed proteins.
  • trans supplementation can be brought about by specialized cells and/or additional viruses producing the needed proteins.
  • Adenoviral vectors lacking all viral protein-coding sequences have been developed. These vectors require supplementation of viral regulatory and structural proteins supplied in trans for packaging and rescue.
  • a second adenovirus carrying genes necessary for virus growth can be used to provide in trans the required supplementation of proteins.
  • helper-dependent adenoviral vector elements and helper adenoviral elements, that enhance the production and isolation of helper-dependent adenoviral vectors.
  • Such elements include a modified packaging signal having low homology to, and preferably less activity than, a wild-type packaging signal, an E4 non-coding segment directly joined to the 5' ITR that confers a selective advantage, and stuffer region(s) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%.
  • the modified packaging signal is preferably used in a helper virus to decrease recombination and generation of the virus.
  • the E4 non-coding segment and the stuffer region(s) are preferably used in a helper-dependent adenoviral vector to provide the vector with a growth advantage over a helper virus.
  • helper-dependent adenoviral vector refers to a viral vector containing the cis elements needed for adenovirus replication and packaging (also referred to herein as viral generation), but lacking the necessary trans elements for adenovirus replication and packaging.
  • the cis elements needed for viral generation is an adenovirus 3' ITR, a packaging signal, and an adenovirus 5' IRT.
  • Trans elements needed for viral generation are the proteins encoded by the El, E2, and E4 and L adenovirus regions.
  • the helper-dependent adenoviral vector lacks nucleic acid encoding for any adenovirus proteins.
  • a helper-dependent adenoviral vector is particularly useful as a vector for delivering genes into cells in vivo.
  • helper virus refers to a virus expressing one or more proteins needed for viral generation of a helper-dependent adenovirus.
  • the helper virus contains the adenovirus 3' IRT, an adenovirus packaging signal, an adenovirus 5' IRT, and encodes for the proteins of the Adenovirus E2 and E4 and L regions.
  • Trans elements not provided for by a helper virus may be provided for by other means such as by a cell. For example, a 293 cell can be employed to supply in trans needed El proteins in conjunction with a helper virus not expressing such proteins.
  • Helper-dependent adenoviral vectors can be produced by co- cultivating with helper viruses in cell lines, wherein the helper virus alone or in combination with the cell line provide the trans functions needed for adenovirus generation.
  • helper virus alone or in combination with the cell line provide the trans functions needed for adenovirus generation.
  • the helper virus alone or in combination with the cell line provide the trans functions needed for adenovirus generation.
  • the helper virus Often, during co-cultivation the helper virus has a selection advantage, such that the mixed population of helper virus and helper-dependent adenoviral vector becomes predominantly helper virus, rather than the desired helper-dependent adenoviral vector. Even when this phenomenon does not occur, the presence of lesser, but significant, amounts of helper viruses still contaminates the helper- dependent adenoviral vector preparation.
  • a first aspect of the present invention describes a nucleic acid molecule comprising an excisable low homology packaging signal cassette.
  • the packaging cassette comprises a low homology packaging signal cassette flanked by a recombinase recognition sequence.
  • the modified packaging signal has low homology to a wild-type adenovirus packaging signal.
  • “Flanked by a recombinase recognition sequence” indicates the presence of a recombinase recognition sequence 3' and 5' of the packaging signal allowing for excision of the flanked nucleic acid containing the packaging signal by a recombinase recognizing the recognition sequence.
  • the recombinase recognition sequence does not need to be immediately 3' or 5' of the modified packaging signal.
  • the 3' and 5' recognition sequence are about 200 bp to about 2,000 bp apart.
  • the modified packaging signal has low homology to a wild-type adenovirus packaging signal and still allows for packaging of the helper virus during its separate production.
  • the modified packaging signal is less efficient than a wild-type adenovirus packaging signal.
  • Low homology refers to a maximum of about 25 bp of contiguous sequence homology (100% sequence identity) between the modified packaging signal and a wild type packaging signal present in an adenovirus, preferably human adenovirus serotype 5.
  • Low homology is determined by aligning the modified packaging signal with wild type packaging signal to obtain a stretch of maximum contiguous homology with sequences present in both the modified and wild type packaging signals.
  • contiguous sequence homology is at most 23 bp.
  • “Less efficient" than a wild-type adenovirus packaging signal indicates that the helper virus has a lower level of packaging when it contains the modified packaging signal than it has when it contains a wild-type packaging signal present in an adenovirus, preferably from a human adenovirus group C serotype, more preferably serotype 5.
  • the helper virus can still be produced at yields of at least 300 PFU/cell in cells lacking a compatible recombinase. Efficiency is preferably determined using titration by end point dilution assay described in the Examples below.
  • modified is not a limitation as to how the packaging signal sequence is produced. Instead, “modified” indicates that the sequence is not a wild-type sequence.
  • Another aspect of the present invention describes an adenoviral helper virus for production of helper-dependent vectors comprising:
  • an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal;
  • an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region.
  • helper-dependent adenovirus vector comprising the following elements (in 5' to 3' order): (a) a 5'- in verted terminal repeat sequence; (b) a packaging signal; (c) one or more heterologous gene expression cassettes; (d) an optionally present E4 non-coding segment conferring a selective advantage; and (e) a 3' ITR; wherein (d) and (e) are located in the distal 400 bp of the adenovirus genome, and wherein the only adenoviral sequences present in the vector are said 5 -inverted terminal repeat, said 3'- inverted terminal repeat, said packaging signal, and said nonexpressed segment of the E4 region.
  • the "E4 non-coding segment” refers to the nonexpressed segment of the E4 region co-localized with the E4 promoter.
  • the segment is adjacent to the 3' ITR and, in human adenovirus serotype 5, is about 300 bp in length.
  • helper-dependent adenovirus vector having a GC content between about 50% and about 60%.
  • the vector comprises in a 5' to 3' direction: (a) a 5' ITR; (b) a packaging signal cassette directly joined to the 3' of the 5' ITR; (c) a first stuffer DNA at least about 1 kb; (d) at least one heterologous expression cassette; (e) a second stuffer DNA at least about 1 kb; (f) an optionally present non-coding E4 segment; and (g) a 3' ITR.
  • Element (f) if present is directly joined to element (g).
  • the virus does not encode one or more adenovirus proteins needed for adenovirus generation and is about 28 kb to about 36 kb. Preferably, the virus does not encode any adenovirus proteins.
  • Another aspect of the present invention describes a cell line infected with a helper virus, a helper-dependent viral vector, or both the virus and the vector.
  • the cell line expresses El proteins and a recombinase, and is infected with a helper virus that (1) contains an excisable low homology signal packaging cassette, and (2) does not express El proteins needed for viral generation.
  • Another aspect of this invention is a method of generating helper- dependent adenoviral gene vectors in a cell line expressing El proteins and Cre recombinase comprising: a) infecting the cell line with a helper-dependent vector comprising: a
  • helper-dependent vector 5' ITR, a packaging signal, at least one heterologous expression cassette, human genomic stuffer DNA and a 3' ITR, wherein the overall size of the helper-dependent vector is between about 28 kb and 36 kb, and wherein no functional adenoviral coding sequences and no bacterial origin of replication or bacterial marker genes are present; b) infecting the cell line with a helper virus comprising: an adenovirus genome having an El region deletion; an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild- type packaging signal; and an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region; and c) obtaining the generated helper-dependent viral vectors.
  • Another aspect of the present invention describes a method of generating helper-dependent adenoviral vectors.
  • the method involves producing a cell comprising: (i) trans functions needed for adenovirus generation and (ii) a helper-dependent adenoviral vector comprising the necessary cis functions needed for adenovirus generation and at least one heterologous expression cassette, wherein the helper-dependent adenoviral vector does not encode for any adenovirus proteins, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%.
  • the cell is used to generate the helper-dependent vector.
  • trans functions can be provided, for example, by a helper virus, the cellular genome, or a combination of both.
  • Another example of a source of trans functions is a plasmid.
  • FIGURE 1 shows the shuffling of fragments of a stuffer to prevent genomic integration of the expression units.
  • FIGURE 2 is a gel showing restriction digests of helper-dependent adenoviral vector DNA after competitive rescues of: a) stkl20gfp-E4-promoter and stkl20gfp; b) stkl20gfp-E4 and stkl20gfp; and c) stkl20gfp-E4-promoter and stkl20gfp-E4.
  • FIGURE 3 is a gel showing restriction digests of helper-dependent adenoviral vectors grown with the helper AdLC ⁇ BHG and a helper of this invention.
  • FIGURE 4 shows the structure of different helper-dependent adenoviral vectors.
  • FIGURE 5 shows autoradiographs of labeled restriction fragments from helper-dependent adenoviral vectors.
  • the figure illustrates the results of competition experiments involving groups of helper-dependent adenoviral vectors from transfection to passage 6.
  • A is a competition of backbones containing the shuffled fragments of cosmid HUMDXS455A (C4).
  • B is a competition of backbones containing part of the HPRT (hypoxanthine guanine phosphoribosyltransferase) genomic gene.
  • C is a competition between 2 backbones one containing part of the HPRT genomic gene the other containing the shuffled fragments of HUMDXS455A (C4).
  • FIGURE 6 is a graph showing the correlation between GC content and helper virus contamination.
  • FIGURE 7 is a graph showing the balance between helper and helper- dependent adenoviral vectors at different time points after infection. The figure demonstrates the replication potential of different helper-dependent adenoviral vectors with the mouse Erytropoetin gene relative to the helper virus.
  • the present invention is particularly useful for obtaining and isolating helper-dependent adenoviral vectors produced using a helper virus.
  • Co-cultivation of a helper-dependent adenoviral vector and a helper virus produces two different particles competing for packaging and results in a mixed viral population.
  • the production of the helper virus in the mixed viral population reduces the amount of helper-dependent adenoviral vector produced, provides a viral contaminant that interferes with applications of the helper-dependent adenoviral vector, and produces a source of nucleic acid that can be recombined with helper-dependent adenoviral vectors
  • production and isolation of helper-dependent adenoviral vectors can be facilitated in different ways.
  • helper-dependent adenoviral vectors containing elements favoring viral generation of the helper-dependent adenoviral vector over a co-cultured helper virus Another way is to employ helper viruses containing elements that can be employed to reduce the ability of the helper virus to grow, and /or recombine with the helper-dependent adenoviral vector.
  • vector employed herein is used in its broadest sense, and is meant to encompass a linear virus which can carry a heterologous gene, and or a plasmid which can contain one or more regions of a virus genome.
  • the term encompasses a helper-dependent adenoviral vector and a helper virus.
  • Adenovirus components present in a helper or a helper-dependent vector may in general be provided from any adenovirus.
  • a human adenovirus serotype is used; more preferably, human adenovirus serotype 1-47; and more preferably, a group C serotype (e.g., serotypes 1, 2, 5, and 6).
  • Preferred group C serotypes are human adenovirus serotype 2 or 5, and more preferably serotype 5.
  • a helper-dependent adenoviral vector preferably contains a heterologous expression cassette.
  • a cassette is made up of a heterologous gene functional coupled to a promoter and regulator elements needed for gene expression, and is particularly useful for introducing nucleic acid into a cell.
  • the introduced nucleic acid preferably encodes for a gene or is able to regulate gene expression.
  • Nucleic acid able to regulate gene expression include ribozymes and antisense nucleic acids.
  • the ability to introduce nucleic acid into a cell using the present invention has different applications such as in gene therapy, in antibody production (e.g., a vaccine) and research to examine the regulation of a gene in vivo or in vitro.
  • Different genes can be introduced into a cell such as Factor VIII, Factor VIX, CFTR, OTC, LDL, VEGF, FGF, EPO, HSV-TK, EL-2, IL-12, p53, HLA-B7, -interferon, and cytosine deaminase.
  • a promoter is a DNA sequence directing the synthesis of RNA through an RNA polymerase.
  • Suitable promoters depend upon the gene and intended use, and may include promoters such as EFl , CMV, chicken ⁇ -actin (Arnold, et al., Nucleic Acids Res 1988 Mar 25;16(6):2411-29), and muscle creatine kinase (Johnson, et al, Mol Cell Biol 1989 Aug;9(8):3393-9 ).
  • the regulatory elements that are present include a ⁇ bosome binding site, a terminator, and an optionally present operatoi. Another preferred element is a polyadenylation signal.
  • Regulatory systems are available to control gene expression, such as GENE-SWITCHTM (Wang, et al, Gene Ther. 1997 May;4(5):432- 41, U.S.
  • Patent No. 5,874,534 and International Publication WO 93/23431 each of which are hereby incorporated by reference herein
  • those involving the tetracychne operator U.S. Patent Nos 5,464,758 and 5,650,298, both of which are hereby incorporated by reference herein.
  • a helper-dependent adenoviral vector is preferably from about 28 kb to about 36 kb to provide for efficient packaging.
  • An adenovirus can accommodate up to about 105% of the wild-type genome and has a lower packaging limit of about 75% of the wild-type genome. (See, Parks, et al, J. Virology 71(4)3293-3298, 1997, hereby incorporated by reference herein.)
  • there are no bacte ⁇ al plasmid-based sequences (such as an o ⁇ gin of replication or bacte ⁇ al marker genes) present in the helper-dependent adenoviral vector.
  • helper viruses and helper-dependent viruses can be achieved based on the guidance provided herein using techniques well known in the art, such as those desc ⁇ bed by Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al, in Molecular Cloning, A laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein Additional descriptions of useful techniques are provided, for example, in the Example section provided below and references such as Mitani, et al, Proc. Natl. Acad. Sci. USA 92:3854-3858, 1995; Fisher, et al, Virology 277:11-22, 1996; Kochanek, et al , Proc.
  • the helper-dependent adenoviral vector and the helper virus can be generated in a wide range of host cells able to be infected by adenovirus.
  • host cells include 293 cells, 911 cells and PERC.6 cells (WO 97/00326 hereby incorporated by reference herein).
  • the employed cell can be constructed to provide for useful proteins such as viral proteins or a recombinase.
  • Helper virus elements useful for facilitating the production and isolation of helper-dependent viruses include a low homology excisable packaging signal cassette and a substituted E3 region.
  • Preferred helper viruses contain both a low homology excisable packaging signal and a substituted E3 gene.
  • the low homology excisable packaging signal cassette contains a modified packaging sequence which fulfills the role of an adenovirus packaging signal, has a low homology to the Adenovirus packaging signal, and is flanked by a 3' and 5' recombinase recognition sequence.
  • the modified packaging signal is less efficient than a wild type packaging signal.
  • Advantages of such low homology excisable packaging signals include: (1) less recombination between the helper virus and helper-dependent adenoviral vector; and (2) when a less efficient packaging signal is used, less packaging of those helper viruses which escaped excision of the packaging signal.
  • Recombinase recognition sequences recombine when acted on by a recombinase that recognizes the sequences, resulting in the excision and circula ⁇ sation of intervening nucleic acid.
  • the recognition sequences should be sufficiently far apart, at least about 180 bp, to allow for the proper positioning of nucleic acid segments being recombined.
  • the recombinase recognition sequence is loxP or frt.
  • the loxP recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Cre recombinase.
  • Cells such as 293 ere cells, stably expressing Cre recombinase can efficiently remove nucleic acid located between loxP recognition sites.
  • the frt recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Flp recombinase.
  • Cells stably expressing Flp recombinase can efficiently remove nucleic acid located between frt recognition sites.
  • Flp-mediated gene modifications are desc ⁇ bed in U.S. Patent No. 5,564,182, hereby incorporated by reference herein.
  • helper-dependent adenoviral vector encourages recombination events between the two, resulting m unwanted changes in the structure of the helper-dependent adenoviral vector or the helper virus, and leading to an increased contamination by helper virus. For example, if the first of a loxP site flanking the packaging signal is removed by homologous recombination within the packaging signal, the resulting helper virus escapes selection in 293cre cells because the Cre recombinase is no longer able to excise the packaging signal.
  • Sequences for different low homology excisable packaging signal cassettes can readily be designed taking into account recombinase recognition sequences and adenovirus wild-type packaging signal sequences using the guidance provided herein.
  • the provided guidance focuses on the adenovirus serotype 5 packaging signal to illustrate the production of a modified packaging signal. Such guidance is applicable to producing other modified packaging signals taking into account other adenovirus serotypes.
  • the wild-type packaging signal of adenovirus serotype 5 is formed by at least seven functional units called A repeats, which are located between nt 230 and nt 380 of the genome.
  • the A elements have the consensus sequence ATTTGNsGC
  • the modified packaging signal of this invention preferably comprises less packaging elements than the wild-type (which has seven elements), preferably from about two to six elements, and more preferably from three to five elements.
  • the modified packaging signal contains only four out of the seven original packaging elements (elements Al to AIV).
  • the four elements are preferably in a modified form with two strong elements (Al and AH) present. The position of the elements relative to each other may be changed, but in preferred embodiments, it is maintained.
  • the eight ambiguous nucleotides of the consensus sequence (ATTTGNsGC; SEQ. ID. NO. 1) within each
  • a element are preferably replaced by sequences taken from a different A element.
  • the eight nucleotides within Al were replaced by those from AV; and the eight nucleotides within AH were replaced by those from AVI.
  • a new element was created between Au and AIII starting 21 bp after AH, by changing the existing nucleotides to the consensus sequence.
  • Two more nucleotides were exchanged within AIV: ATTTTGTGTT (SEQ. ID. NO. 2) was changed to ATTTTGTTGT (SEQ. ID. NO. 3).
  • ATTTTGTGTT SEQ. ID. NO. 2
  • ATTTTGTTGT SEQ. ID. NO. 3
  • One embodiment of a synthetic packaging signal is given in SEQ. ID. NO. 4.
  • Desired nucleic acid sequences can be produced using different techniques including those involving the creation of nucleic acid mutations and nucleic acid synthesis techniques. Examples of such techniques are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al, in Molecular Cloning, A laboratory Manual, 2 nd Edition, Cold Sp ⁇ ng Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein.
  • the helper virus also contains an insertion element.
  • the insertion element does not contain a promoter or encode for a protein when present in the helper virus. More preferably, the insertion element is non- coding repeat-free human DNA devoid of splice signals.
  • non-cod g sequences as an E3 insertion element, preferably human non-gene sequences, provides for more efficient viral generation than that observed with E3 msertional elements encoding for bacte ⁇ al protein described by Parks, et al, Proc. Natl. Acad. Sci. USA 95.13565-13570, 1996 (see Table 1, infra.)
  • intron sequences are prefe ⁇ ed because such sequences do not contain hidden splice signals that could interfere with correct splicing of the fiber message.
  • the size of the insertion element (preferably between about 2800 and 3500 bp, more preferably about 2900 bp) is chosen to avoid the generation of potentially harmful wild-type viruses while maintaining a virus size close to wild-type size.
  • the recombined adenoviral vector which also contains the E3 insertion element would have a genome size of approximately 38350 bp (108.1% of wild-type size). Because this size exceeds the packaging limit of Adenovirus 5, no viable virus would be produced.
  • the insertion element may be from virtually any source.
  • a 2900 bp insert was taken from human chromosome 1 lql3, starting 13340 bp upstream of STS marker US 1337 (available from Genethon) and inserted into the Xba I site at position (nt) 28593 (referring to Ad5 wild-type).
  • the insertion element and its insertion point in E3 should be chosen to avoid any reduction in synthesis of the fiber protein. Such a reduction is likely to occur if the E3 region is modified because synthesis of fiber protein is dependent on co ⁇ ect splicing of RNA from signals located in this region.
  • viruses containing the insert grow to titers similar to those obtained with viruses containing an unchanged E3 region.
  • Helper-dependent adenoviral vector elements useful for facilitating the production and stability of helper-dependent adenoviral vectors include an E4 non- coding segment that confers a selective advantage to a virus and stuffer region(s) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%.
  • the helper-dependent adenoviral vector contains an E4 non- coding segment conferring a selective advantage to the vector; and stuffer region(s) providing the vector with a GC content of about 50% to about 60% and an overall size of about 28 kb to about 36 kb.
  • there are no bacterial plasmid-based sequences such as an origin of replication or bacterial marker genes
  • Stuffer sequences are used to provide a helper-dependent adenoviral vector with an overall size of about 28 kb to about 36 kb.
  • a helper-dependent adenoviral vector containing only a heterologous expression cassette, adenoviral ITR's and a packaging signal may be quite small, in general about 5-10 kb, depending on the size of the heterologous expression cassette. Because such a small virus does not package efficiently stuffer sequences are added to provide a final size of about 28 kb to about 36 kb.
  • the stuffer provides the vector with a GC content of about 50% to about 60%.
  • Stuffer sequences used in gene therapy applications should not contain active genes or be recognized as foreign in a human cell.
  • a preferred stuffer sequence replicates as well as the adenovirus itself, is not recognized by the host as foreign DNA and does not lead to chromosomal integration of the transgene.
  • Genomic DNA of mammalian origin or even more preferably of human origin, are preferred stuffer sequences for gene therapy applications to avoid eliciting an immune response.
  • stuffer based on human genomic DNA is identical to chromosomal sequences in a human target cell and could allow for insertion of the heterologous expression cassette into the host genome by homologous recombination. This phenomenon could have a potentially dangerous effect and should be prevented.
  • the contiguous stuffer sequence can be inte ⁇ upted, the individual fragments reversed and the expression units inserted at the specific breakpoints. (See for example, Figure 1.)
  • the transgene is not flanked by regions which would support homologous recombination.
  • potentially unstable genetic elements such as retrovirus LTR as well as genomic repeats (e.g., MIR, and ALU) should avoided or removed to prevent rea ⁇ angements during amplification.
  • the GC content of the virus Another consideration is the GC content of the virus.
  • the GC content that stuffer sequences provide to a helper-dependent adenoviral vector has a major impact on yield and purity of the virus.
  • Competition experiments between different viruses as well as individual analysis revealed a correlation between the GC content of a virus and growth properties.
  • the GC content is preferably between about 50% and about 60% and even more preferably between 52% and 57% which is close to the GC of wild-type adenovirus.
  • advantageous growth properties are believed to be based on more efficient generation of a virus with a higher GC content.
  • stuffer sequences are provided such that the heterologous gene cassette(s) that are present are at least about 1.0 kb from the helper-dependent viral vector 3' end and 5' end, more preferably at least about 2.0 kb, and more preferably at least about 4.0 kb.
  • high AT rich segments such as those provided in the hprt fragment (59% AT) may be unfavorable even with a viral vector having an overall high GC content.
  • a "high" AT is above about 55% AT.
  • the E4 region of adenovirus type 5 occupies about 3000 bp at the right end of the genome. Transcription from the E4 promoter is directed from the right end toward the center. Seven polypeptides are produced from open reading frames (ORFs) located within the region with the first ORF starting at nucleotide (nt) 406 from the right end of the virus.
  • ORFs open reading frames
  • helper-dependent adenoviral vector generally is favored in co-cultivation with the non-recombined helper-dependent adenoviral vector and dominates in vector preparations.
  • this recombined helper-dependent adenoviral vector cannot be used as a helper-dependent vector for gene therapy because it contains functional E4 genes. Expression of those E4 genes in a host cause an immune rejection of vector infected cells by the host.
  • E4 sequences located left of this element have no additional impact on virus strength. This is of importance because all genes (open reading frames, orfs) are located in the left part of the E4 region.
  • another aspect of this invention is a ' s-acting sequence located next to the 3' ITR (right virus end) which is preferably incorporated into the helper-dependent adenoviral vector because it supports amplification of the helper-dependent adenoviral vector.
  • Helper-dependent adenoviral vectors based on the vector stkl20 (which contains only a 5' ITR, a 3' ITR and packaging signal as its viral-based nucleotides, as described in Morsy et al, 1998, Proc. Natl Acad. Sci.
  • the discriminating fragment was found 5-10 fold stronger for stkl20gfp-E4-promoter than for stkl20gfp, and at least 4 fold stronger for stkl20gfp-E4 than for stkl20gfp (see Figures 2a and 2b).
  • the discriminating fragments are of equal strength for stkl20gfp-E4-promoter and stkl20gfp-E4 in each rescue (see Figure 2c). Therefore an E4 element supporting helper-dependent adenoviral vector growth is present in stkl20gfp-E4 promoter and stkl20gfp-E4 and must be entirely located in the right part of the E4 region (between nucleotide -400 from the right end and the 3' ITR).
  • the E4 element allows for more effective replication of the virus or it confers improved packaging and stability.
  • Our results indicate that the E4 segment does not effect replication. If infection is started with equal amounts of helper and helper-dependent adenoviral vectors, the ratio between helper and helper-dependent genomes inside the cell at the end of the production cycle (48 hours) is identical. However, 3-fold more helper-dependent adenoviral vectors were packaged for the genome containing the E4 segment compared to contaminating helper genomes. Thus, it appears that the E4 segment supports effective packaging.
  • E. coli based on the recF pathway
  • Competent cells of E. coli strain BJ 5183 were transformed with a plasmid vector containing the adenovirus genome and a fragment from the shuttle vector. This fragment contains the sequence to be inserted flanked on both sides by sequences homologous to the vector. In order to select against re-transformation of the vector without modification, it was linearized close to the insertion point. DNA isolated from individual colonies of BJ5183 was retransformed into the strain XL2 (Stratagene) and high yield plasmid preparations were obtained. Correct clones were determined by restriction analysis and sequencing.
  • Virus Rescue lO ⁇ g helper plasmid was digested with Pad and transfected by calcium phosphate coprecipitation into a 6 cm dish of subconfluent 293 cells. Directly after transfection cells were overlaid with ⁇ MEM/ 10% FCS/ 0.8 % Seaplaque agarose. 20-100 plaques appeared after 7-9 days. Plaques were taken 12 days after transfection and used to reinfect cells. After two intermediate passages the cell lysate was used to infect NUNC-cell factories (NUNC). Cells were harvested 48 hours after infection and virus was released by three freeze/thaw steps steps (-70°C /37°C).
  • Virus DNA was cut with Hind III or other restriction endonucleases, restriction fragments were labeled using ocP33dATP or dCTP and Klenow polymerase and separated in 0.7% agarose gels. The gel was dried and exposed to film.
  • 10 6 cells are lysed inlOmM Tris-HCl pH 7.5, lOmM EDTA, 0.5% SDS and treated with 100 ⁇ g/ml Proteinase K overnight at 58°C.
  • the lysate was extracted with Tris saturated phenol, phenol/chloroform/ isoamylalcohol (24:24: 1) and chloroform, precipitated with two volumes of ethanol, washed extensively with 70% ethanol and resuspended in TE.
  • 96 well plates were seeded with 1x104 293 cells in lOO ⁇ l per well 24 hours before the assay. Each virus stock was diluted 103 to 108- fold. Dilutions were used to infect 24 wells each (50 ⁇ l/ well). Positive wells were scored by cytopatic effect after 14 days and the virus titer was calculated based on two dilutions with intermediate numbers of positive wells taking the dilution factor into account.
  • Quantitative PCR Real time quantitative PCR was used to determine the relative amounts of helper and helper-dependent adenoviral vector.
  • Two specific target sequences were selected present either in all helper viruses (Ad5 sequences from 11358-11456) or in all helper-dependent adenoviral vectors based on stkl20 (bp 12037-12176) (Morsy et al, 1998, Proc. Natl. Acad. Sci. USA 95:7866-7871).
  • a plasmid containing both target sequences was constructed and used as one standard for both amplifications.
  • a set of forward primer, reverse primer and probe (which is located between the primers and contains a fluorogenic reporter and a quencher) have been selected. Separation of quencher and reporter generated a fluorogenic signal during the logarithmic phase of PCR amplification which was plotted versus cycle number. The plot was used to calculate template concentration based on the software of the 7700 system (ABI).
  • Helper viruses for the helper-dependent system were made based on Ad5.
  • the plasmid pAdEl-E3+ (containing the entire Ad5 genome with a complete deletion of the El region) was modified in two regions: i) between the left ITR and the promoter of protein IX; and ii) in the E3 region.
  • E a linker containing cloning sites
  • F 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).
  • a second shuttle vector, "ploxpack”, containing the wild type packaging signal instead of the synthetic one was constructed. It contained: A) nt 1-195 of Ad 5 linked to a unique Pac I site immediately before nt l;
  • F 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).
  • the second shuttle vector was generated by cloning the 4.9kb Xhol fragment from Ad5 wild-type (Ad5wt) DNA (nt 24797-29791) into pUC.
  • Ad5wt Ad5 wild-type DNA
  • a 2900 bp fragment of human DNA located on chromosome 1 lql 3 starting 13340 bp upstream of STS marker 1 IS 1337 (Genethon) was obtained by PCR and inserted into the Xbal site at nt 28593 (Ad5wt) in either orientation. This fragment contains part of inton 2 of the human LRP5 gene.
  • a strategy based on homologous recombination between plasmids in
  • E. coli (Chartier, et al., 1996 J. Virol 70: 4805-4810, which is hereby incorporated by reference) was used to introduce those sequence elements into plasmid pAd5El-E3+ containing a complete Ad5 genome with a deletion of El. The procedure allowed for insertion of those elements at exactly the positions described for the shuttle vectors. The flanking Ad5 derived sequences were completely maintained.
  • pAdlspLIl Helper 14 — has synthetic packaging signal, intron of LRP5 in E3;
  • Plasmids containing the Ad 5 genome with the described modifications were digested with Pad to release the virus genome from the plasmid and transfected into 293 cells by calcium phosphate coprecipitation. Resulting virus plaques were used to reinfect cells. Amplified virus stocks were characterized by restriction and sequence analysis.
  • helper viruses as well as a El deficient adenovirus (dl70-3) (Bett et al. Proc. Natl. Acad. Sci. USA 91:8902-8906 which is hereby incorporated by reference) taken as control, were grown in 10 layer NUNC cell factories, purified in CsCl Gradients and dialyzed. The virus concentration (particle concentration) was determined by spectro-photometric analysis (OD260)- L5 x 106 cells (293 or 293 cre) were infected in a 6 cm plate with 100 particles per cell. CPE was apparent in all plates after 48 hours. Cells and medium were harvested, frozen and thawed 3x and the titer was determined in an end point dilution assay. The number of infectious viruses produced per cell was calculated and the results are shown in Table 1.
  • AdLC8BHG101uc (Parks et al, 1996, Proc. Natl Acad. Sci. USA 93:13565-13570, which is hereby incorporated by reference) by at least 20 fold.
  • the growth of the helper viruses in contrast to dl70-3 is well controlled in 293cre4 cells. These cells release only 1-2 infectious particles/cell. The rate of suppression is therefore dramatically increased for the new helper viruses Viruses without or with the insert in E3 in either o ⁇ entation do not differ much in virus yields, thus, the chosen E3 insert did not have a major impact on virus growth.
  • a characte ⁇ zed stock (known particle titer and helper contamination) of a helper-dependent adenoviral vector containing the human OTC gene was used to infect 293cre cells.
  • Cells were infected with 700 particles of the helper-dependent adenoviral vector and 100 particles of one of the helper viruses per cell After 48 hours cells were collected, virus was released through 3 freeze/thaw steps and pu ⁇ fied by Cs banding Particle titers were determined by spectrophotomet ⁇ c analysis (OD260)- Virus DNA was extracted and analyzed by rest ⁇ ction digests ( Figure 3).
  • helper contamination was measured using quantitative PCR based on TaqmanTM technology. Contamination was similar between the helper AdLC8BHG101uc and the helper LPLI1 containing the o ⁇ g al packaging signal, whereas the amplification supported by helper LSPLIl containing the modified packaging signal resulted in a lower contamination (0.29%) (Table 2). This is expected because yields of virus grown in 293 cells are lower than those of an identical virus containing the normal packaging signal. Therefore helper virus which would escape cre-selection should still have a selective disadvantage.
  • Constructions are based on plasmid stkl20 (Morsy et al, 1998, supra) containing a CMV promoter driven gfp (green florescent protein) gene within the stuffer sequences at a unique Swal site (stkl20gfp).
  • the 3' ITR 130 bp was extended to 400 bp from the right end of the Ad5 genome. This fragment contains the complete E4 promoter region. However, no adenovirus coding sequences are present.
  • the resulting adenoviral vector is stkl20gfp-E4-promoter.
  • Genomes for stkl20gfp and stkl20gfp-E4-promoter were released from the respective plasmids and cotransfected with a circular plasmid containing the entire helper genome into 293 cre cells at a 1:1:2 ratio followed by infection with helper virus at moi 1.
  • the mixture of viruses was amplified up to passage 7 where the viruses were Cs-purified. Five independent experiments were carried out.
  • group A began with the ligation of a 3498 bp from fragment from pSTK120 (which comprised of the bacterial sequences origin of replication and ampicillin resistance gene, the right and left ITRs and the packaging signal) ligated with a multiple cloning site linker (SEQ. ID. NOs. 6 and 7) containing Malawi! sticky ends and cloning sites Eagl, Asd, Bgi ⁇ , Notl, Xbal, Mlul, EcoRl and Kasl. This clone is named "pITRF".
  • DNA from the region encompassed within HUMDXS455A (Genebank Accession # L31948) used as stuffer DNA was amplified from human genomic DNA in four segments of about 4.5kb each using the following pairs of primers: SEQ. ID NOs. 8 and 9 (PCR 1) SEQ. ID NOs. 10 and 11 (PCR2) SEQ. ID NOs. 12 and 13 (PCR3) SEQ. ED NOs. 14 and 15 (PCR4)
  • PCR1 product was Klenow filled in and then digested with Ascl. It was ligated directly into the Ascl/EcoRV of pITRF to produce "Construct 1".
  • PCR2 product was digested with Notl/Ascl and cloned into the
  • PCR3 product and Construct 2 were digested with Mlul/Notl (double digest) and ligated to produce "Construct 3".
  • the PCR4 product was first cloned into a TA cloning vector. To avoid inefficient direct cloning for very large plasmids homologous recombination in E. coli BJ 5183 was used to insert PCR4 and additional genomic fragments. The technique requires a small shuttle vector containing the region which overlaps with the insertion point in the large plasmid. This shuttle vector was constructed as follows: 2 kb of the junction over PCR3 and vector sequence was amplified using Construct 3 DNA.
  • the PCR fragment was Klenow filled in and then digested with Sapl enzyme and ligated into PABS helper-dependent-3.0, a small plasmid vector (KanR) cut with ⁇ coRI followed by a fill in reaction using Klenow and a digestion with Sapl. Homologous recombination between Construct 3 and the shuttle resulted in Construct 4 (C4).
  • the helper-dependent adenoviral vector part of C4 is 20kb in length and has 3 unique rest ⁇ ction enzyme sites to incorporate 3 different transgenes (Ascl, Notl and Swal).
  • a CMV promoter d ⁇ ven gfp gene was inserted into the Notl site of C4 by homologous recombination.
  • a shuttle vector was built containing 2 kb of C4 overlapping the Notl site (pNot).
  • the gfp expression unit was inserted by blunt end cloning into Notl and a fragment of this vector was used for homologous recombination with C4. Because a single Notl site is also present in the gfp expression unit, the gene can later be replaced by other genes using this rest ⁇ ction site.
  • the gfp gene is located at a junction between 2 inverted genomic fragments which prevents insertion into the host genome by homologous recombination which would be likely, if the gene was inserted withm a contiguous sequence.
  • a second shuttle based on pABSHD-3 was constructed containing 2 kb of C4 including the 3' ITR and unique Kasl, Sail, Bglll and HindHI sites for insertion.
  • the E4 promoter was directly cloned as a Hind HI/ Sail fragment into this shuttle vector.
  • Stuffer DNA fragments AFO, HSU, ER1, and ER2 were amplified from human genomic DNA by PCR Expand Kit (Boeh ⁇ nger) and cloned into the Topo-blunt vector (Invitrogene).
  • AFO and HSU were inserted individually as Sail fragments, the ER1 and ER2 fragments were inserted sequentially after Kasl/ Bglll and BglH/ Sal digests. Recombination in E. coli BJ 5183 lead to the final backbones C4HSU (SEQ. ID. NO. 16), C4AFO (SEQ. ID. NO. 17), and C4ER ( Figure 4).
  • This step removes a part of the hprt region from the vector which was most frequently lost during adenoviral vector propagation.
  • This vector "STKSEgfp" is 19.4 kb in length and misses about lOkb of genomic sequence required for propagation as a helper- dependent adenoviral vector.
  • Fragments AFO and HSU were cloned after Sail digestion into the Sal I site of shuttle vector pSHl-3ITR which contains part of the HUMDXS455A - fragment in STK 120 and the 3' end of Ad5 starting from nt -400 containing the E4 promoter and the 3' ITR.
  • the fragments ERl and ER2 were inserted step by step into the same vector as Sail /BamHl and BamHl/Bgl ⁇ fragments.
  • the 3 resulting different shuttle vectors were recombined with STKSEgfp linearized with Pad to generate HXAFO, HXHSU and HXER ( Figure 4).
  • helper-Dependent Adenoviral Vectors Cells from subconfluent plates were washed with PBS, trypsinized and seeded to 6 well plates (Costar) at 3 x 10 ⁇ cells per well in 1.5ml medium. Genomes for helper-dependent adenoviral vector were released from the respective plasmids by Pme I digestion and 2 ⁇ g of DNA were cotransfected with a 2 ⁇ g circular plasmid containing the entire helper genome per well. A 24 hour transfection was followed by infection with helper virus at 1 moi. Forty-eight hours later cells were lysed by 3 freeze/thaw cycles and the lysate was used to infect 106293cre cells together with Helper virus at MOI 5.
  • helper-dependent adenoviral vector is isolated by CsCl banding and analyzed by restriction digestion.
  • Passage 6 is also used to infect a large scale preparation (1x10 layer Nunc cell factory).
  • Example 5 Competition Between Helper-Dependent Adenoviral Vectors Virus rescue in a competition experiment was performed using the procedures described above and, instead of DNA from a single vector plasmid, a equimolar mixture of 3 vectors was used. Three mixed rescues were carried out: one with HXAFO, HXHSU and HXER; one with C4AFO, C4HSU and C4ER; and the third with HXHSU and C4HSU. No rearrangements were observed in either the individual or the mixed rescues.
  • the gfp gene was replaced in STK120, C4AFO and C4HSU by the in mice immunologically inert mEPO gene. Replacement was carried out using the shuttle pNot by homologous recombination. Viruses were rescued as described above. Large scale amplifications were carried out in Nunc cell factories and the virus was purified as described above.
  • helper-dependent adenoviral vector Since the size of the helper-dependent adenoviral vector and the helper differ only slightly, the purification procedure does not remove helper virus from the preparation. Such a physical separation was not intended to allow for a replacement of gradient based by chromatography based purification in large scale.
  • DNA was extracted from purified virus and the content of helper virus relative to the amount of helper-dependent adenoviral vector was determined by TaqmanTM quantitative PCR. We observed a correlation between the helper content and the GC content of the helper-dependent adenoviral vector (see Figure 6). The experiment with Construct C4HSU has the lowest helper content (about 0.18%) compared to a helper content of about 1% for stkl20.
  • Example 7 Analysis Of The Replication Potential Of Different Helper-Dependent Adenoviral Vectors
  • Cells were infected with 100 part/cell of helper and 300 part/cell of the helper-dependent adenoviral vector. Three hours after infection 1/3 of the cells was harvested and washed with PBS. Another portion was harvested at 42 hours and a third fraction at 48 hours. Cellular DNA was extracted as described above and the relative amount of helper and helper-dependent adenoviral vector DNA was determined by TaqmanTM PCR ( Figure 7).
  • Figure 7 shows that if the rescue is started with an excess of helper- dependent adenoviral vector, the ratio of helper-dependent/helper is maintained and even slightly increased for C4HSU, whereas it is strongly decreasing for STK 120 and slightly decreasing for C4AFO. This shows that the nature of the backbone has a strong impact on the speed of replication. Whereas the Adenovirus genome coevolved with the adenovirus polymerase, the helper-dependent adenoviral vector sequence was artificially selected. Therefore a less efficient replication is expected. However, the most important determinant seems to be the GC content of the helper- dependent adenoviral vector.

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Abstract

Cette invention concerne des éléments vecteurs adénoviraux dépendant d'un assistant ainsi que des éléments d'adénovirus assistant qui favorisent la production et l'isolement de vecteurs adénoviraux dépendant d'un assistant. De tels éléments comprennent un signal d'encapsidation présentant une faible homologie avec un signal d'encapsidation de type sauvage, et de préférence une activité réduite par rapport à ce dernier, un segment non codant E4 lié directement à 5' ITR qui confère un avantage sélectif, et une ou des régions stuffer qui fournissent un vecteur adénoviral dépendant d'un assistant avec une teneur en GC de 50 à 60 % environ. Le signal d'encapsidation modifié sert de préférence à diminuer la recombinaison et la production de virus. Le segment E4 non codant et la ou les régions stuffer sont utilisés de préférence dans un vecteur adénoviral assistant-dépendant d'un assistant pour conférer à ce vecteur un avantage de croissance sur le virus assistant.
PCT/US2000/002405 1999-02-04 2000-01-31 Amelioration du systeme vecteur dependant d'un assistant-dependant pour la therapie genique WO2000046360A1 (fr)

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US09/890,836 US7132277B1 (en) 2000-01-31 2000-01-31 Helper dependent vector system for gene therapy
JP2000597420A JP2002535986A (ja) 1999-02-04 2000-01-31 遺伝子治療用の改良ヘルパーディペンデントベクター系
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WO2002002788A2 (fr) * 2000-06-29 2002-01-10 Genzyme Corporation Procedes de production de vecteurs adenoviraux dependants d'un auxiliaire
WO2002027007A2 (fr) 2000-09-25 2002-04-04 Regents Of The University Of Michigan Production de vecteurs viraux
WO2005112541A3 (fr) * 2004-05-20 2006-03-23 Proyecto Biomedicina Cima Sl Vecteur hybride adenovirus-alphavirus destine a l'administration de maniere efficace et a l'expression de genes therapeutiques dans des cellules tumorales
WO2007125146A1 (fr) * 2006-04-28 2007-11-08 Universitat Autonoma De Barcelona Procédé de production de vecteurs adénovirus destinés a la thérapie génique et séquences d'adn utilisées à ces fins
US7803365B2 (en) * 2002-12-02 2010-09-28 Biovec, Llc Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases
US8048410B2 (en) 2002-12-02 2011-11-01 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002788A3 (fr) * 2000-06-29 2003-01-03 Genzyme Corp Procedes de production de vecteurs adenoviraux dependants d'un auxiliaire
WO2002002788A2 (fr) * 2000-06-29 2002-01-10 Genzyme Corporation Procedes de production de vecteurs adenoviraux dependants d'un auxiliaire
US7820441B2 (en) 2000-09-25 2010-10-26 The Regents Of The University Of Michigan Production of viral vectors
WO2002027007A2 (fr) 2000-09-25 2002-04-04 Regents Of The University Of Michigan Production de vecteurs viraux
WO2002027007A3 (fr) * 2000-09-25 2003-02-06 Univ Michigan Production de vecteurs viraux
US10167485B2 (en) 2000-09-25 2019-01-01 The Regents Of The University Of Michigan Production of viral vectors
US9453240B2 (en) 2000-09-25 2016-09-27 The Regents Of The University Of Michigan Production of viral vectors
EP2336339A3 (fr) * 2000-09-25 2011-09-14 The Regents of the University of Michigan Production de vecteurs viraux
US8637313B2 (en) 2000-09-25 2014-01-28 The Regents Of The University Of Michigan Production of viral vectors
US7803365B2 (en) * 2002-12-02 2010-09-28 Biovec, Llc Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases
US8420075B2 (en) 2002-12-02 2013-04-16 Biovec, Llc Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases
US9353383B2 (en) 2002-12-02 2016-05-31 Biovec, Llc Vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
US8048410B2 (en) 2002-12-02 2011-11-01 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
US8236300B2 (en) 2002-12-02 2012-08-07 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
US8242095B2 (en) 2002-12-02 2012-08-14 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
US8367056B2 (en) 2002-12-02 2013-02-05 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
US7850957B2 (en) 2004-05-20 2010-12-14 Proyecto De Biomecdicina Cima, S.L. Adenovirus/alphavirus hybrid vector for the effective administration and expression of therapeutic genes in tumour cells
ES2292271B1 (es) * 2004-05-20 2009-02-16 Proyecto De Biomedicina Cima, S.L. Un vector hibrido adenovirus-alfavirus para la administracion eficaz y expresion de genes terapeuticos en celulas tumorales.
ES2292271A1 (es) * 2004-05-20 2008-03-01 Proyecto De Biomedicina Cima, S.L. Un vector hibrido adenovirus-alfavirus para la administracion eficaz y expresion de genes terapeuticos en celulas tumorales.
WO2005112541A3 (fr) * 2004-05-20 2006-03-23 Proyecto Biomedicina Cima Sl Vecteur hybride adenovirus-alphavirus destine a l'administration de maniere efficace et a l'expression de genes therapeutiques dans des cellules tumorales
WO2007125146A1 (fr) * 2006-04-28 2007-11-08 Universitat Autonoma De Barcelona Procédé de production de vecteurs adénovirus destinés a la thérapie génique et séquences d'adn utilisées à ces fins

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