WO1999053085A2 - Regulation selective de la production d'adenovirus - Google Patents

Regulation selective de la production d'adenovirus Download PDF

Info

Publication number
WO1999053085A2
WO1999053085A2 PCT/US1999/008294 US9908294W WO9953085A2 WO 1999053085 A2 WO1999053085 A2 WO 1999053085A2 US 9908294 W US9908294 W US 9908294W WO 9953085 A2 WO9953085 A2 WO 9953085A2
Authority
WO
WIPO (PCT)
Prior art keywords
packaging
adenovirus
repressor
coup
dna
Prior art date
Application number
PCT/US1999/008294
Other languages
English (en)
Other versions
WO1999053085A3 (fr
Inventor
Patrick Hearing
Susanne I. Schmid
Philomena H. Ostapchuk
Ece Erturk
Original Assignee
The Research Foundation Of State University Of New York
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Research Foundation Of State University Of New York filed Critical The Research Foundation Of State University Of New York
Priority to NZ502175A priority Critical patent/NZ502175A/xx
Priority to IL13351099A priority patent/IL133510A0/xx
Priority to EP99917543A priority patent/EP0994958A2/fr
Priority to AU35636/99A priority patent/AU770005B2/en
Priority to JP55211099A priority patent/JP2002506355A/ja
Publication of WO1999053085A2 publication Critical patent/WO1999053085A2/fr
Publication of WO1999053085A3 publication Critical patent/WO1999053085A3/fr

Links

Classifications

    • 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
    • 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

Definitions

  • This invention relates to the field of adenovirus vectors and the regulation of viral particle production.
  • One area of application is in the field of DNA delivery systems.
  • Adenovirus is a common human DNA virus that naturally infects the airway epithelia as well as other tissues in the body.
  • Adenovirus (“Ad”) is a particularly useful virus as a human DNA delivery system for a number of reasons.
  • Ad has a wide host cell range, and recombinant Ad vectors have been used to efficiently infect multiple cell types in culture and in animals.
  • Adenoviruses have been shown to infect a variety of tissues in animal studies including liver, kidney, muscle, respiratory, endothelial and nervous system.
  • Ad has the ability to efficiently infect non-dividing differentiated cells in the animal, a major target for DNA delivery applications.
  • adenovirus is a relatively benign human virus that is associated with mild disease, and importantly is not associated with the development of any human malignancy.
  • Adenovirus-based vectors offer several unique advantages, including tropism for both dividing and non- dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts.
  • the cloning capacity of an adenovirus vector is about 8-10 kb, resulting from the deletion of certain regions of the virus genome dispensable for virus growth, e.g., E3, deletions of regions whose function is restored in trans from a packaging cell line, e.g., El, and its complementation by 293 cells (Graham, (1977)), and deletion of E2b (Amalfitano, et al., (1998)) and E4 (Krougliak, et al.(1995); Brough, et al . (1996)) as well as the upper limit for optimal packaging which is about 105% of wild-type length.
  • Adenovirus DNA encapsidation occurs in a polar manner from left to right and relies on a cis-acting packaging domain located between approximately nt 200- 380 (Daniell et al . (1976); Hammerskjoeld et al . (1980);
  • Ad5 packaging domain consists of at least seven redundant, albeit not functionally equivalent, elements termed A repeats I through VII (Graeble et al. (1990); Graeble et al . (1992) ) .
  • a major goal in DNA delivery systems is to create a viral vector that lacks all viral coding sequences, and only contains DNA of interest for delivery purposes plus minimal viral DNA sequences required for growth and production of the virus.
  • a helper virus is required, but selection against contamination of the virus stock with the helper virus (wild type virus) must be imposed.
  • the only system described to date to selectively repress packaging of an adenovirus helper virus is the excision of the packaging domain using the CRE-LOX system. This system reduces packaging of the helper virus 100- or
  • RCA replication competent adenovirus
  • RCA is the generation of wild type, infectious adenovirus via the recombination between two different viruses within an infected cell. RCA arises primarily through homologous recombination between two viruses coinfected in a cell between overlapping homologous DNA sequences, or between virus DNA and viral DNA integrated into host chromosomes in certain complementing cell lines used to grow such a virus.
  • One object of the present invention is the identification of a specific control element which mediates the function of the adenovirus packaging domain. This control element operates through binding of one or more trans-acting proteins.
  • Another object of the present invention relates to the identification of a minimum packaging signal which can direct adenovirus packaging.
  • Yet another object of the present invention relates to adenovirus vector constructs having a selectively regulated packaging function.
  • Another object of the present invention relates to the use of adenovirus vectors with a regulated packaging function in a DNA delivery system.
  • Yet another object of the present invention relates to repressor-mediated control of adenovirus o particle production containing binding sites for such repressors and the use of vectors containing such binding sites for DNA delivery.
  • the present invention relates to adenovirus vectors containing a minimum packaging signal for producing adenovirus virions.
  • a minimum packaging signal for producing adenovirus virions Of special importance is the presence of a CG dinucleotide located downstream of a TTTG sequence within each of the packaging elements. Spacing between the consensus segment 5'-TTTG-3' and the 5'-CG-3' segment located downstream is preferably between 1 and 12 nucleotides. Alternatively, it may be preferred to configure the consensus segments so that
  • the adenovirus vector of the present invention may contain a packaging element consisting of 5' -TTTGN 8 CG-3 ' which represents a minimal sequence necessary for adenovirus packaging.
  • 25 sequence is preferably present in multiple copies.
  • One type of minimal packaging sequence is an "A repeat", which contains a consensus sequence.
  • a repeat sequences are shown in Table 1.
  • Another aspect of the present invention relates to novel vectors containing the minimum packaging sequences which can be selectively regulated.
  • One such embodiment provides an adenovirus vector g ⁇ r containing minimum packaging sequences and repressor sites, such as COUP-TF or lac repressor sites.
  • Such vectors are selectively packaged in the absence of the repressor.
  • the repressor sites may flank the packaging sequence, may be embedded into the packaging sequence or may alternate the packaging sequence.
  • Such vectors may contain one type of repressor site or combinations of different repressor sites.
  • the present invention also relates to a cellular DNA binding protein, called COUP-TF, which binds to adenovirus DNA packaging sequences. It has been found that over-expression of COUP-TF in cells infected with adenovirus specifically represses virus production; in particular, virus packaging. COUP-TF preferentially binds to certain packaging elements.
  • adenovirus vectors of the present invention may contain one or more COUP-TF binding sites.
  • Adenovirus vectors of the invention may contain a combination of COUP-TF binding sites and minimal packaging sequences. These elements can be used to selectively regulate packaging of such viruses.
  • the present invention relates to a method of regulating adenovirus packaging comprising the steps of obtaining an adenovirus vector containing a repressor binding site, propagating this vector in the absence of the repressor and repressing packaging of said vector in the presence of COUP-TF.
  • a method may be carried out in one cell line.
  • the propagating step may be carried out in a first cell line and the repressing step may be carried out in a second cell line.
  • the repressor may be endogenous to the cell line or exogenously provided at the DNA or protein level.
  • the present invention provides adenovirus vectors that package the virus using one or more COUP-TF binding sites or, for example, one or more A repeats.
  • the present invention provides a selective system to control the packaging of an adenovirus vector.
  • the system can be designed to allow efficient packaging of one adenovirus vector while inhibiting packaging of a different vector in the same infected cell by using viruses with different packaging sites and/or COUP-TF binding sites in conjunction with COUP-TF over-expression.
  • Yet another aspect of the present invention provides a method of treating a patient through the administration of a heterologous gene that is expressed in the patient or a DNA fragment that is itself therapeutically active in the patient.
  • This gene or DNA is delivered to the patient via an adenovirus vector which is prepared for administration using a regulatable adenovirus vector of the present invention.
  • the present invention also relates to P- complex, an activity involved in adenovirus packaging.
  • P-complex appears to contain TATA-binding protein ("TBP") and TAF172 and is useful in production or packaging of viral particles.
  • TBP TATA-binding protein
  • TAF172 TATA-binding protein
  • P-complex interacts with the minimum packaging signal of adenovirus.
  • FIG. 1 depicts the adenovirus type 5 packaging domain.
  • A A schematic representation of the left end of the adenovirus type 5 genome. Nucleotide positions are indicated by numbers. The inverted terminal repeat (ITR) is represented by a gray box. Viral packaging repeats are termed A repeats I to VII (arrows) . The EIA transcriptional start site is indicated by an arrow, and enhancer elements I and II are designated as EIA enhancer.
  • B The packaging repeat consensus motif. Shown is an alignment of A repeats I, II, V and VI.
  • Nucleotides comprising the bipartite consensus motif for A repeats I, II, V and VI are boxed and enlarged. The consensus motif is shown at the bottom (5" -TTTGN 8 CG-3 ' ) .
  • the positions of AV and AVI are shown by horizontal lines above the sequence. Nucleotides identical between all subgroups are indicated by vertical lines.
  • FIG. 2 depicts the functional hierarchy among different packaging repeats.
  • a schematic representation of left-end sequences of wild-type adenovirus is shown at the top (as per Fig. 1A) .
  • a repeats Al, All, AV and AVI are represented by boxes of distinct shading.
  • the mutant viruses contain a deletion between nucleotides 194 and 814, and the insertion of 6 copies each of AVI (194/814:AVI6) , All (194/814 :AII6) and Al (194/814 :AI6) , a dimerized copy of AV, AVI and AVII (194/811 :AV-AVII2) or 12 copies of AVI (194/814 :AVI12) .
  • Mutant virus yields in the single infections are expressed as fold-reduction relative to that of the wild-type virus.
  • the results from the coinfection experiments are expressed as fold-reduction in packaged mutant DNA relative to packaged wild-type DNA.
  • NV virus was not viable.
  • ND packaged viral DNA was below the level of quantitation.
  • FIG. 3 depicts a cellular complex (P complex) which interacts with adenovirus packaging elements.
  • a gel mobility shift competition experiment is presented. Radio labelled probe (AV-VII dimer) 293 nuclear extract and nonspecific competitor DNA (polydldC) were incubated in the absence (lanes 1 and 24) or presence (lanes 2 to 23) of competitor oligonucleotides . P-complex DNA binding activity is indicated by an arrow. Increasing amounts of specific competitor oligonucleotides are indicated, and represent a 40- and 200-fold molar excess of A repeats relative to the probe. The competitors are named according to the A repeats they represent. An LS was appended when the TTTG consensus motif in the oligonucleotide was mutated. A CG was appended when the CG consensus dinucleotide was mutated.
  • FIG. 4 depicts P-complex and adenovirus DNA packaging.
  • the left terminus of the adenovirus genome is schematically represented with ITR and packaging domain denoted by boxes.
  • Trans-acting components binding ITR and packaging sequences are identical in the model on the left, whereas different factors interact with the respective sequences in the model on the right as indicated by circles.
  • FIG. 5 depicts the scheme used for P-complex purification.
  • FIG. 6 depicts the binding of COUP-TFI to minimal packaging domains.
  • Gel mobility shift assays were performed using COUP-TFI synthesized by in vi tro translation.
  • a hexamer of A repeat VI (lanes 1-9) and a hexamer of A repeat I (lanes 10-18) were used as radiolabelled probes.
  • Unprogrammed reticulocyte lysate (Unprog) or increasing amounts of COUP-TFI-programmed lysate (COUP) was used in binding reactions.
  • P preimmune serum
  • ⁇ -COUP antiserum
  • FIG. 7 depicts multimerized oligonucleotides corresponding to A repeats Al and AVI used to construct recombinant viruses.
  • a dimeric oligonucleotide sequence is shown to simplify the schematic diagram.
  • the potential COUP-TF binding sites in the oligonucleotides are indicated by arrows.
  • Perfect or 4-out-of-5 nucleotide matches to the COUP-TF consensus sequence are shown as closed arrowheads; 3-out-of-5 nucleotide matches to the COUP-TF consensus site are shown as open arrowheads.
  • Perfect, or nearly-perfect, COUP-TF binding sites with a 1 base spacing are found in multiple locations in the AVI oligonucleotide repeat, but not in the Al oligonucleotide repeat.
  • FIG. 8 depicts a scheme for growth of a "gutted” adenovirus gene therapy vector and the specific repression of packaging of a helper virus needed to grow the "gutted” virus.
  • the "gutted” adenovirus lacks viral coding regions and contains the inverted terminal repeats (ITRs) required for DNA replication and a hexamer of A repeat I (for example) to direct viral DNA packaging.
  • the remainder of the recombinant adenovirus vector is available for the insertion of large DNA segments (28 to 36 kbp) .
  • the helper virus carries all of the wild type adenovirus genome and the packaging domain is replaced with multimerized copies (12) of A repeat VI.
  • the helper virus is grown without COUP-TFI overexpression to allow for the high level production of the helper virus.
  • cells that overexpress COUP-TFI are coinfected with the "gutted” adenovirus and the helper virus.
  • the helper virus allows for the production of Ad early and late gene products for complementation in trans of the "gutted” adenovirus.
  • the packaging of the DNA genome of the helper virus is specifically repressed by COUP-TFI overexpression, while packaging of the genome of the "gutted” adenovirus is not repressed since its packaging elements do not bind COUP-TFI .
  • Fig. 9 depicts the specific repression of packaging of a "designer" adenovirus vector by expression of COUP-TF.
  • A The growth of adenovirus USFO was measured without or with expression of COUP-TF. 293 cells were cotransfected with USFO DNA plus increasing concentrations of empty expression vector (CMX) or an expression vector for high level production of COUP-TF (CMX-COUP-TF) . Virus yield (log virus yield) was measured by plaque assay on 293 cells. COUP-TF expression had a minimal effect of production of the USFO virus.
  • B The growth of adenovirus USFO+AVI 12 was measured, as described in (A) .
  • COUP-TF expression specifically repressed production of the "designer" virus USFO+AVI 12 .
  • the maximum level of repression of packaging of USFO+AVI 12 by COUP-TF expression was 400- fold.
  • C Western blot analysis of adenovirus late protein expression without or with COUP-TF expression. 293 cells were cotransfected with USFO DNA without or with expression of COUP-TF. Adenovirus late protein fiber and penton were quantified by Western blot using specific antibodies. The results show COUP-TF expression has a minimal effect on adenovirus late gene expression.
  • FIG. 10 depicts synthetic oligonucleotides that contain different adenovirus packaging repeats designed with specific repressor binding sites that either overlap the packaging A repeats or are placed between packaging A repeats.
  • A The sequence of the wild type AV-AVII oligonucleotide. A dimeric copy of this oligonucleotide efficiently directed packaging in a recombinant virus (Fig. 2) . A repeats V, VI and VII are indicated and the consensus packaging repeats are encircled.
  • the AV-AVII oligonucleotide is modified (underlined nucleotides) to create a high affinity binding site for the adenovirus-induced E2F-E4-6/7 protein complex overlapping A repeats V and VI (binding site indicated by inverted arrows) .
  • the AV-AVII oligonucleotide is modified (underlined nucleotides) to create a high affinity binding site for the E. coli lac repressor overlapping and adjacent to A repeat V (binding site indicated by inverted arrows) .
  • FIG. 11 (A) Western blot showing lac repressor expression in 293 cells and (B) gel mobility shift assay showing lac repressor protein expressed in 293 cells binds to the AV-AVII + lac site shown in Fig. IOC.
  • Fig. 12 depicts the specific repression of packaging of a "designer" adenovirus vector by expression of lac repressor. The growth of adenovirus
  • AV-VII+lac was measured without or with expression of lac repressor.
  • 293 cells were cotransfected with AV- Vll+lac DNA plus increasing concentrations of empty expression vector (CMX) or an expression vector for high level production of lac repressor (CMX+lac repressor) .
  • Virus yield (log virus yield) was measured by plaque assay on 293 cells. Lac repressor expression specifically repressed production of the "designer" virus AV-VII+lac. The maximum level of repression of packaging of AV-VII+lac by lac repressor expression was 20-fold.
  • the present invention relates to regulation of adenovirus packaging. Both cis- and trans-acting elements are described. These elements control adenovirus packaging, and as such, their selective use in adenovirus vectors for DNA delivery can reduce the danger of producing RCA in viral preparations and in patients .
  • the present invention is directed to regulatable adenovirus vectors. These new vectors have specific packaging sequences and are regulated so that production of viral particles is controlled.
  • the vector design also increases the safety of recombinant adenovirus vectors for use as DNA transfer vehicles by reducing the potential for RCA.
  • the adenovirus vectors of the present invention may be derived from any known adenovirus serotype.
  • the A repeats used as minimum packaging sequences may also be derived from any adenovirus serotype.
  • Several example A repeats and their similarity between serotypes are illustrated in Figure 1C '
  • One aspect of the invention identifies that a COUP-TF binding site acts as an active site for repression of adenovirus packaging.
  • another aspect of the invention identifies a complex, termed P- complex which is involved in packaging.
  • Packaging is a critical function of the adenovirus for production of viral particles.
  • One important use for a regulated adenovirus vector is in the field of DNA delivery for therapeutic applications which uses a viral vector to deliver genes or DNAs of interest to a patient in need of such treatment.
  • DNA delivery system refers to a system of delivering a DNA to a patient.
  • a DNA may contain a gene encoding a protein whose expression in the patient may provide a therapeutic benefit.
  • proteins may, for example, act as a treatment for a disease or condition, or may stimulate an immune response, such as a vaccine.
  • Gene therapy is one such DNA delivery system.
  • the DNA of interest may not encode a protein yet may provide a benefit to the patient.
  • a DNA may act as a antiviral agent or may transcribe into an RNA which may act as an antisense therapeutic or antiviral agent.
  • the present invention also relates to the identification of a minimum adenovirus packaging signal.
  • a minimal packaging sequence of 5 ' -TTTGN 8 CG-3 ' has been identified. Although eight nucleotides are preferred to separate the left portion of the packaging consensus element (i.e., 5'-TTTG-3') from the right portion (i.e.,
  • this spacing may vary 1 to 12 nucleotides.
  • the packaging element may be inserted into the left or right end of the adenovirus vector, preferably within 600 nucleotides from either end. More preferably, this minimal sequence is present at the left end of the genome and is present in multiple copies.
  • Another consensus sequence comprises 5'-ATTTGN 8 CG-3 * and provides a strong packaging signal in adenovirus vectors. Two copies of this minimal packaging sequence are sufficient to ensure packaging. More than two copies enhance virus packaging. However, any number of this sequence can be inserted into the virus to ensure particle production.
  • Multimerized refers to multiple copies of an element (i.e. packaging or repressing) . These elements may be present in single units or in multimers, which preferably means 2-36 repeats and more preferably 2-12 units or elements.
  • One form of the minimal packaging element is an "A repeat", which is derived from adenovirus. Representative A repeats are set forth below in Table 1:
  • adenovirus vectors that contain minimal packaging domains have been developed consisting of multimerized oligonucleotide sequences in place of the normal packaging domain. Additionally, these new adenovirus vectors may contain deletions of viral DNA sequences from the left end of the genome which allow for augmented insertion of foreign DNA sequences in the context of DNA delivery vectors. Up to 400 nucleotides can be deleted from the left end of the genome and be replaced with the minimum packaging sequences defined herein to produce a vector with an increased capacity to carry foreign DNA.
  • packaging oligonucleotide repeats in different individual viral vectors allows for the selective repression of packaging of one adenovirus vector, but not another adenovirus vector, in cells coinfected with both viruses.
  • the latter scenario is important in the design of a vector capable of selective packaging for use in DNA delivery systems, and the repression of packaging of a helper virus needed to grow the adenovirus vector.
  • the vectors of the present invention are useful in DNA delivery systems to help curb the production of replication competent adenovirus (RCA) , a virus that is dangerous and potentially toxic to a patient receiving it during patient administration. This is due to the fact that two distinct viruses can be made with entirely distinct, and non-overlapping packaging domains.
  • a virus eg. gutted gene therapy virus 1
  • gutted gene therapy virus 1 may contain a hexamer of A repeat
  • helper virus virus #2
  • virus #2 may contain a dimer of A repeats V, VI and VII or a multimer of AVI in an inverted orientation.
  • both viruses carry functional packaging domains, but overlap homologous recombination is greatly minimized since different packaging sequences and DNA orientations are used.
  • a target for homologous recombination does not exist in the packaging domain.
  • the use of different packaging domains in the two viruses greatly minimizes the possibility of recombination between the two viruses to generate RCA.
  • one or two copies of a DNA segment containing packaging A repeats V, VI and VII direct packaging.
  • a single copy of the segment functions for packaging.
  • This type of packaging sequence contains a series of different repeats and is referred to as a natural packaging domain.
  • the second type of packaging sequence contains a single type of A repeat which when multimerized functions efficiently for packaging.
  • This segment is referred to as a synthetic packaging element.
  • Vectors of the present invention may contain a combination of natural and synthetic packaging elements.
  • the present invention approach to DNA delivery vector design preferably uses a "gutted" adenovirus vector whereby most or all of the viral genes are removed.
  • "gutted” vector approach There are two advantages with "gutted” vector approach. First, little or no viral proteins are produced following infection that normally elicit an immune response. Second, such a virus is capable of carrying very large gene inserts for gene therapy applications. For example, the dystrophin gene for treatment of muscular dystrophy is 14,000 bp in length necessitating a vector with very large insert capacity. Also, the Factor VIII gene for treatment of hemophilia A is greater than 7000 bp. Additionally, it may be preferable to use tissue-specific regulatory sequences to produce tissue-specific expression of a gene. This requires increasing the insert capacity in a vector, because many tissue-specific promoters contain several thousand base pairs.
  • genes and/or DNA segments may be carried by adenoviral vectors.
  • genes include; interleukin-2 (Haddada, et al. (1993)) p53 (Harris, et al. (1996)); l-antitrypsin (Jaffe, et al.(1992), cystis fibrosis transmembrane conductance regulator (CFTR) (Rosenfeld et al . , (1992)), and clotting factor VIII (Connelly, et al . (1995)).
  • interleukin-2 Haddada, et al. (1993)
  • p53 Harris, et al. (1996)
  • l-antitrypsin Jaffe, et al.(1992)
  • cystis fibrosis transmembrane conductance regulator CFTR
  • clotting factor VIII Connelly, et al . (1995)
  • the recombinant adenovirus of the present invention is preferably a "gutted vector" and contains adenovirus sequences at the left and right termini required for DNA replication and two or more copies of the minimal packaging sequence to direct viable DNA packaging.
  • the remainder of the recombinant adenovirus vector is available for insertion of large DNA segments (up to 36,000 base pairs).
  • a helper adenovirus is needed to grow such a "gutted" vector in order to produce all of the viral proteins that are missing in the "gutted vector".
  • helper virus a virus necessary for replication of the viral construct
  • a helper virus is designed to contain a COUP-TF binding site and is first allowed to grow productively in the absence of COUP-TF, then is blocked from being packaged by the presence of COUP-TF.
  • the viral growth is carried out in a cell line which does not express COUP-TF and the packaging is blocked by the addition of COUP-TF protein.
  • the viral growth is carried out in a cell line lacking COUP-TF (Qiu, et al . (1997)) and the packaging repression step is accomplished by transfer of the virus into cells expressing COUP-TF. In this way, helper virus can be used to propagate the adenovirus vector yet not be present in the final viral preparation.
  • Another important aspect of the present invention relates to gene therapy vectors that use adenovirus minimal packaging sequence, 5'- TTTGN 8 CG -3'. (See Provisional patent application no. 60/081,867, incorporated herein by reference) .
  • adenovirus vector design of the present invention utilizes a packaging/repressor system.
  • adenovirus vectors are constructed with alternating oligonucleotides containing the minimal packaging sequence and binding sites for a repressor.
  • a lac repressor site can be inserted between packaging sequences.
  • the lac repressor is a high affinity binding repressor not found in eukaryotic cells.
  • Another example of such a system embeds one or more repressor sites within a packaging domain.
  • Yet another example of a packaging/repressor system flanks a packaging domain with surrounding repressor binding sites.
  • This system may have one or a series of repressor binding sites to the left of a minimal packaging domain and another set of repressor binding sites to the right of a packaging domain.
  • a virus which contains minimal packaging sequence and repressor binding sites such as, for example, lac repressor sites, can be grown in cells not expressing the repressor, and then packaging can be selectively repressed in cells expressing high levels of the repressor.
  • the present invention also provides vectors containing a packaging sequence in combination with the COUP-TF repressor binding sites whose packaging capability can be selectively controlled.
  • such vectors may have a packaging sequence containing a dimer of A repeats V, VI.
  • These packaging domains may also contain a COUP-TF repressor site as well as signals sufficient to allow efficient packaging.
  • Such vectors allow packaging in the absence of COUP-TF repressor, but inhibit packaging in the presence of COUP-TF.
  • cells that overexpress COUP-TFI can be infected with the therapeutic adenovirus vector containing one type of packaging element (for example, multiple copies of A repeat I) and the helper Ad containing a different type of packaging element (for example, multimerized copies of A repeat VI) .
  • the packaging of the helper virus will be specifically suppressed by COUP-TFI overexpression, while packaging of the genome of the adenovirus gene therapy vector will not be repressed.
  • a conditional system for repression of packaging is designed into the vector so that a helper virus can be grown to high levels under non-repression conditions, and then specific repression of the helper virus packaging accomplished when used to complement growth of the therapeutic virus vector.
  • lac repressor binding sites are embedded within a minimal packaging domain.
  • a packaging domain may be engineered to contain a lac repressor binding site embedded within the A repeat V, VI and VII packaging domain.
  • the virus can then grow in the absence of lac repressor expression while repression of packaging (e.g. a helper virus) is observed with high level lac repressor expression.
  • the virus can then grow in the absence of lac repressor expression while packaging is repressed when lac is expressed.
  • E2F transcription factor binding sites are embedded within a minimal packaging domain.
  • a high affinity binding site for a DNA binding protein is embedded within a minimal packaging domain with the ability to selectively "activate” the repressor.
  • the cellular transcription factor (E2F) and an adenovirus protein (E4-6/7) which induces the cooperative and stable binding of E2F to an inverted binding site provide the packaging/repressor system of this vector.
  • a high affinity E2F inverted binding site is inserted within a minimal packaging domain containing, for example, A repeats V, VI and VII.
  • this mutant virus In the absence of 6/7 protein expression (this mutant virus is completely viable) , E2F binding to the packaging region is weak and thus repression is weak. In the presence of the E4-6/7 protein, E2F binding is stable and with high affinity. Thus, binding of the bona-fide packaging factor is repressed and packaging of the virus is blocked.
  • binding sites for other repressors such as, for example, lambda repressor or Tet repressor, can be employed in the design of adenovirus vectors of the present invention. Other potential repressor sites can be employed and will be readily known to the skilled artisan.
  • Multimers of different A repeats are able to direct packaging of viral DNA but at different efficiencies (Schmid and Hearing, 1998) .
  • Any of the A repeats may serve as a minimal packaging sequence.
  • these A repeats are used as multimers in a packaging element.
  • a hexamer of A repeat II can also be used in the present invention, having a moderate activity.
  • a hexamer of A repeat VI is also a packaging element, albeit a weak element.
  • a repeat VI when utilized as a multimer, preferably a 12-mer, efficiently directs packaging.
  • One embodiment of the present invention relates to vector constructs containing multimers of the A repeat VI packaging signal which is a high affinity binding site for COUP-TF binding. Such a vector construction can be regulated through selective expression of COUP-TF.
  • COUP-TF binds to adenovirus packaging sequences.
  • Overexpression of COUP-TF resulted in a 10, 000- fold decrease in the production of infectious adenovirus. This effect was, at least in part, due to repression of the adenovirus major late promoter ("MLP") which directs the synthesis of adenoviral late mRNAs and thus viral late proteins.
  • MLP adenovirus major late promoter
  • COUP-TF binds to a specific DNA sequence in the MLP that overlaps the binding site for the activating transcription factor called USF (Sawadogo and Roeder, 1985) .
  • COUP-TF is a known repressor of eukaryotic transcription promoter activity (Cooner et al., 1992; Tsai and Tsai, 1997).
  • P- complex was found to interact with cellular complexes in the viral packaging machinery (Schmid and Hearing, 1998) .
  • a direct correlation is seen between the binding affinity of P-complex for different A repeats in vi tro and the ability of the respective fragments to support DNA packaging in vivo .
  • the TTTG, but not the CG, packaging consensus half site is critical for P-complex interaction.
  • the P-complex binds to core replication sequences in the inverted terminal repeat (ITR) .
  • ITR inverted terminal repeat
  • the cellular P-complex activity by virtue of its ability to interact with both packaging and core replication sequences, constitutes a trans-acting link between viral DNA replication and encapsidation.
  • the binding of a cellular transcription factor, COUP-TF, to minimal segments of the viral packaging domain was also detected. Its binding affinity does not correlate with viral DNA packaging in vivo, but rather repression thereof.
  • Cellular P-complex is a bona-fide adenovirus packaging component. This complex appears to contain a TATA binding protein (TBP) and a second protein called TAF172 (Timmers et al . 1992, Taggart et al . 1992). P- complex binding is inhibited by ATP and magnesium. Complex formation is observed on all minimal packaging domains that exhibit functional activity in vivo .
  • TBP TATA binding protein
  • TAF172 TAF172
  • Complex formation is observed on all minimal packaging domains that exhibit functional activity in vivo .
  • the affinity of the P complex for the different multimeric A repeats in vi tro correlates well with the ability of the respective cis-acting sequences to support viral DNA packaging in vivo .
  • Al and AV-VII constitute strong P complex binding sites and they confer maximal packaging activity in vivo .
  • the most preferred P-complex binding sites comprise a hexamer of Al and a dimer of AV, AVI and AVII.
  • AVI is noted as a weak binding site for P complex in vi tro, and it serves as a particularly weak packaging domain in vivo .
  • the Ad packaging consensus motif is a bipartite sequence with a conserved AT-rich and a GC-rich half site (5 ' TTTGN 8 CG-3 * ) (Schmid, et al. (1997) ) .
  • TBP-TAF172 The identification of the DNA binding activity of P complex as containing TBP-TAF172 has important implications for the development of "designer" adenovirus vectors for repression of packaging. For example, using the viruses depicted in Fig. 8, a gutted gene therapy vector may be generated that binds P complex/TBP-TAF172 poorly using mutations in the AT-rich binding site that reduce TBP binding to DNA in the helper virus packaging sequences. Additionally, so- called “altered-specificity" TBP mutants may be used in the present invention (Strubin and Struhl, 1992) . Such mutations produce TBP protein having altered specificity for binding to certain DNAs.
  • adenovirus vectors with altered specificity P complex/TBP-TAF172 binding sites may be constructed to conditionally repress packaging of a helper virus.
  • the helper virus contains the altered specificity TGTA binding site in place of the AT-rich part of the A repeat; the virus can be successfully propagated when altered-specificity TBP is provided in cells, and packaging of the helper virus repressed when grown in cells lacking the altered-specificity TBP.
  • Other manipulations of the P complex/TBP-TAF172 binding site and/or manipulations of the DNA binding proteins can be made by the skilled artisan toward the same goal.
  • This complex corresponds to the P-complex detected in our gel mobility shift assays since it exhibits binding specificity for both packaging and ITR sequences.
  • the AT-rich packaging consensus half site is implicated in the initial recognition of A- repeats by packaging factors. Perhaps the CG-rich half site and proteins bound to it are involved in secondary events like capsid recognition or insertion of the viral DNA into the capsid. It is noteworthy that the 8 bp spacing, or one helical turn of the DNA, which separates the AT-rich and the CG-rich consensus half site is important for DNA encapsidation in vivo . This may reflect the need for a physical interaction between components of the P-complex and CG-bound unidentified components, to allow for the timing and/or coordination of successive steps in adenovirus DNA packaging.
  • Ad5 dl309 the parent for all the viruses described in this report, is a phenotypically wild type virus that contains a unique Xbal cleavage site at 3.8 map units (Jones, et al. (1979)).
  • Plasmid pElA-194/814 contains the left end Ad5 Xbal fragment (nt 1-1339) with a deletion between nt 194 and 814 and a unique Xhol restriction site at the junction of the deletion.
  • a head-to-tail hexamer of an oligonucleotide containing A repeat VI (5 ' -TCGACCGCGGGGACTTTGACC-3 ' :
  • pBR-194/814 and pBR-53/814 have sequences between nt 194 and 814 and nt 53 and 814 deleted.
  • a monomer and dimer of viral sequences is located between nt 334 and 385 which contain AV, AVI, and AVII was cloned into the 194/814 deletion.
  • a dimer of the nt 334 to 385 fragment as well as 12 head-to-tail copies of an oligonucleotide containing AVI (5'- TCGACCGCGGGGACTTTGACC-3 ' : 5 ' -TCGAGGTCAAAGTCCCCGCGG-3 ' ) were cloned into the 53/814 deletion in either orientation. All mutations were verified by nucleotide sequence analysis. The recombinant plasmids were subsequently rebuilt into intact viruses by the method of Stow (1981). Viruses were amplified and titered on 293 cells. Mutant viruses were screened by restriction analysis of viral DNA obtained from infected 293 cells by the Hirt procedure (Hirt (1967)), and all insertions were verified by nucleotide sequence analysis of viral DNA using PCR-based sequencing.
  • Virus stocks were generated by three freeze-thaw cycles of infected cell lysates and titered by plaque assays on 293 cells. Virus infections were performed at a multiplicity of infection (MOI) of 3 PFU per cell for 1 h at 37°C. Cells were then washed twice with tris-buffered saline solution and overlayed with fresh medium.
  • MOI multiplicity of infection
  • virus yield Determination of virus yield and packaging efficiency. Both assays were performed as described previously (Schmid, et al . (1997)). For the determination of virus yield in a single infection, infected cell lysates were prepared 48 h post-infection and the amount of infectious virus was determined by plaque assays on 293 cells. Packaging efficiency of the mutant viruses was tested in a coinfection of 293 cells with both mutant and wild-type dl309 virus. Forty-eight hours post-infection, one half of the cells was used to isolate total nuclear DNA, the other half was used for the preparation of viral DNA from purified virions.
  • the data presented for virus yield in the single infections and the data for packaging efficiency based on coinfection experiments represent the averages of at least three independent experiments.
  • Extract preparation and gel mobility shift assays Nuclear extracts were prepared by the method of Dignam and Roeder (1983) , and dialyzed overnight against 20 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.5), 100 mM NaCl, 10% glycerol, 5 mM MgCl , 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (DB-100) . The dialysate was cleared by centrifugation at 25,000 x g for 20 minutes.
  • HEPES N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • the binding reaction was carried out in a total volume of 20 ⁇ g for 1-2 hr at room temperature in 40 mM HEPES pH 7.5, 70 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 10 ⁇ g/ml BSA and 4% Ficoll.
  • the complexes were resolved electrophoretically at 10 V/cm on a 3.5% 30:1 (acrylamide: bisacrylamide) polyacrylamide gel in 0.5x TBE (25 mM Tris pH 8.3, 25 mM boric acid, 0.5 mM EDTA) at 4°C.
  • a monomer of AV-VII is: 5'-
  • a repeat II (5 1 - TCGACCGAGTAAGATTTGGCC-3 ' : 5 ' -TCGAGGCCAAATCTTACTCGG-3 ' ) and A repeat V (5 ' -TCGACCGCGTAATATTTGTCC-3 ' : 5'-TCGAGGACAAATATTACGCGG-3') were used as multimeric competitors.
  • Packaging repeat competitor fragments designated LS have the underlined nucleotides shown above in Al, All, AV, AVI, AV-VI mutated into the sequence 5'GTGCAG-3' (only the upper strand is indicated) .
  • the italicized CG dinucleotide in the AV competitor was replaced by an AT in the competitor fragment designated CG.
  • the competitor oligonucleotide representing ITR sequences 1-13 was used in monomeric form and was identical to the one used for probe preparation.
  • the monomeric ITR 10-22 competitor oligonucleotide contains sequences between Ad nt 10-22 flanked by Xhol/Sall linkers. Quantitation of oligonucleotide competitors was performed spectrophotometrically. The amount of specific competitor DNA added per binding reaction is indicated in the text as -fold molar excess of binding sites present in the competitor relative to binding sites present in the probe. This definition, however, is based on the assumption that one binding site (located between nt 1-13) is present in monomeric ITR fragments and that six binding sites are present in hexameric packaging repeat fragments.
  • Proteins separated by SDS-polyacrylamide gel electrophoresis were transferred to nitrocellulose, and probed with different antibodies (rabbit polyclonal anti-COUP, anti-fiber and anti-penton antisers, monoclonal antibody M45) . Proteins were visualized using a secondary horseradish peroxidase- conjugated antibody and chemiluminescence as recommended by the manufacturer (Amersham) .
  • Minimal adenovirus packaging domains Minimal adenovirus packaging domains .
  • Adenovirus packaging elements are functionally redundant, but in spite of this redundancy, different elements are not functionally equivalent with respect to each other. Elements I, II, V and VI constitute the most functionally dominant A repeats (Graeble et al. (1990); Graeble et al . (1992); Schmidt et al. (1997)).
  • a repeat VI as an independent cis-acting unit (Schmid, et al . (1997)).
  • a hexamer of A repeat VI in place of the packaging domain yields a viable virus, although the mutant is reduced >100-fold in growth compared to wild-type.
  • Such a mutant is under strong evolutionary pressure for the amplification of packaging elements since revertants with significantly improved growth were found to evolve by amplification of preexisting copies of A repeat VI.
  • a fragment containing A repeats V-VII functions efficiently to direct packaging and these A repeats did not amplify upon virus propagation (Fig. 2; Schmid, et al. (1997)).
  • Sequences flanking the packaging domain are identical in both of these mutant viruses (a deletion of sequences between nt 194 and 814) . This raises the question of whether there is a hierarchy of importance among the four most- dominant A repeats with A repeat VI as a functionally less dominant element, or alternatively, whether a combination of different elements supports packaging better than only one type of A repeat.
  • viral mutants were constructed that contain multimers of individual A repeats inserted into a 194/814 deletion background (Fig 2) (Schmid and Hearing, (1998)).
  • the packaging domain was replaced by a hexamer in the forward orientation of AVI, All and Al, respectively.
  • the parent virus was nonviable (described Schmid, et al . (1997)), and lacking any functional packaging elements. Insertion of a hexamer of AVI, All and Al into the 194/814 deletion background rescued virus viability, albeit to different extents.
  • a multimer of A repeat VI in place of the packaging domain resulted in a virus that exhibited a more than 100-fold reduction in growth in a single infection relative to wild-type virus.
  • DNA packaging in a coinfection with wild-type virus was nondetectable.
  • a hexamer of repeat I supported viral growth in a single infection and DNA packaging in a coinfection better than A repeat II, with a reduction in growth of 4-fold versus 20-fold in the o single infection and in packaging efficiency of 2-fold versus 5-fold in the coinfection, respectively.
  • HeLa cell pellets were obtained from the National Cell Culture Center (Minneapolis, MN) . All procedures were performed at 4°C. Nuclear extract was prepared by the method of Dignam et al. (1983), dialyzed into buffer DB- 100 (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 20% glycerol, 0.1 mM EDTA, 0.5 mM PMSF, 0.25 mM benzamidine, 1.0 mM DTT), and the dialysate centrifuged at 25,000 x g for 20 minutes. Buffer DB is the same buffer but lacks NaCl.
  • Nuclear extract was applied to a heparin-agarose column (10 mg protein/1 ml heparin-agarose) equilibrated in DB-100, the column was washed with DB-100, and bound proteins were eluted with a linear NaCl gradient (0.1 M- 0.6 M) in DB.
  • Fractions containing P complex activity were identified using a gel mobility shift assay with a DNA probe consisting of a dimer of A repeats V-VII (as per Fig. 3). The P complex peak eluted at 0.42 M NaCl.
  • the NaCl concentration was diluted to 0.05 M using DB, and the P complex pool was applied to a phosphocellulose Pll column (8 mg protein/1 ml Pll) equilibrated in DB- 0.05, the column was washed with DB-0.05, and bound proteins were eluted with a linear NaCl gradient (0.05 M-0.6 M) in DB. The peak of P complex activity eluted at 0.13 M NaCl.
  • the P complex pool was diluted to 0.1 M NaCl using DB, and applied to an SP-Sepharose column (8 mg protein/1 ml SP-Sepharose) equilibrated in DB-100.
  • the column was washed with DB-100, and bound proteins eluted with a linear NaCl gradient (0.1 M-0.6 M) in DB.
  • the peak of P complex activity eluted at 0.20 M NaCl.
  • the P complex pool was diluted to 0.1 M NaCl using DB and protease inhibitors aprotinin and leupeptin were added to 1 ⁇ g/ml to all buffers from this point on.
  • the P complex pool was applied to a Q-Sepharose column (8 mg protein/1 ml Q-Sepharose) equilibrated in DB-100.
  • the column was washed with DB-100, and bound proteins eluted with a linear NaCl gradient (0.1 M-0.6 M) in DB.
  • the peak of P complex activity eluted at 0.28 M NaCl.
  • the P complex pool was diluted to 0.1 M NaCl using DB, and NaP0 4 was added to 10 mM.
  • the P complex pool was applied to a hydroxy-apatite column (5 mg/protein/1 ml hydroxy-apatite) equilibrated in DB-100+ 10 mM NaP0 4 .
  • the column was washed with DB-100 + NaP0 4 , and bound proteins eluted with a linear NaP0 4 gradient (10 mM - 250 mM) in DB-100.
  • P complex activity was pooled with a final purification of -1000-fold.
  • a cellular complex interacts with adenovirus packaging elements.
  • Minimal packaging domains defined in vi vo were used as probes for gel mobility shift assays for the detection of trans-acting packaging components. Since such components could be viral and/or cellular in origin, we initially carried out binding studies with both uninfected and Ad-infected 293 cell nuclear extracts. Infections were performed using either wild-type Ad dl309 or a temperature-sensitive virus, tsl9, defective for virus assembly when grown at the restrictive temperature (Williams, et al . (1971)).
  • Extracts from tsl9-infected cells were tested in view of the fact that packaging factors may be encapsidated with wild-type adenovirus and consequently not present in nuclear extracts used for in vi tro binding studies. At no point did we detect any difference between complex formation using nuclear extracts from infected or uninfected cells, and therefore, all experiments presented below were performed with extracts from uninfected cells.
  • FIG. 1 shows the results from a gel mobility shift assay in which this fragment was used as a probe and incubated with uninfected 293 cell nuclear extract for the detection of interacting proteins.
  • lanes 1 and 24 (+ ) no specific competitor was added, whereas a 40- and 200-fold molar excess of competitor oligonucleotides were added to the binding reactions resolved in lanes 2 to 23.
  • the specific competitor fragments are indicated above the autoradiography and represent different multimeric A repeats, either in the wild-type or mutated configuration (see Materials and
  • a slow migrating complex termed the P-complex (indicated by an arrow) was formed on the AV-VII probe (lanes 1 and 24), which disappeared upon self-competition (lanes 2 and 3), but not when the TTTG half-site of the packaging element consensus motif was mutated in A repeats V and VI of the competitor oligonucleotide (lanes 4 and 5) .
  • a cellular binding activity termed P-complex, interacts specifically with various packaging elements in a gel mobility shift assay, in perfect correlation with data obtained in vivo with mutant viruses containing minimal packaging domains. Integrity of the AT-rich, but not the CG-rich, part of the packaging consensus motif is critical for this interaction.
  • P complex interacts with viral core origin sequences.
  • P complex binding activity was bound to bind to sequences derived from the left terminus of the adenovirus genome (Schmid and Hearing, 1998) .
  • a repeat sequences Al hexamer probe or AV to VII dimer probe
  • the binding of P complex to A repeat sequences was efficiently competed by an oligonucleotide containing left en ITR sequences from nucleotides 1 to 13, but not by an oligonucleotide containing ITR sequences from nucleotide 10 to 22.
  • P complex bound efficiently to a DNA probe containing ITR sequences from nucleotides 1 to 13, and this binding was efficiently competed by wild type A repeat oligonucleotide competitors, but not by A repeats with mutation in the TTTG consensus motif.
  • the data show that P complex not only binds to packaging A repeats, but also to the very terminus of the adenovirus genome (nucleotides 1 to 13) .
  • the binding of P complex to the packaging domain and left terminus of the adenovirus genome followed by P complex protein-protein interaction may result in looping of the intervening DNA sequences.
  • the competition experiments are consistent with one or two possibilities for P complex binding activity.
  • P complex may contain one DNA binding activity that recognizes both packaging A repeats as well as the left terminus of the adenovirus genome (which is AT-rich but does not have a consensus A repeat sequence) .
  • Second Fig. 4 o
  • P complex may consist of two distinct but interacting activities whereby one DNA binding activity binds the consensus A repeat sequence and the second DNA binding activity binds to the AT-rich left terminus of ⁇ r the adenovirus genome.
  • COUP-TF interacts with adenovirus packaging elements.
  • COUP-TF binds to the consensus sequence 5'-GGTCA-3' when situated as a direct or inverted repeat, with a preferred spacing of 1 base pair, and represented as perfect or imperfect versions of the consensus binding site. These binding sites overlap A repeat VI (5' -GGACTTTGACC-3' ; the COUP-TF inverted repeat is underlined, and AVI is in bold) , only the upper strand is indicated with the COUP half sites underlined and AVI indicated in bold case. Other A repeats contain similar sequence motifs, albeit with less resemblance to the dimeric COUP consensus.
  • COUP-TF binds to A repeats when synthesized in vi tro
  • helper virus in Fig. 8 containing the USF-0 mutations in the MLP was generated.
  • the salient features of the vector are: mutations of
  • This new "designer" helper virus vector is termed USF-0 + AVI 12 .
  • a repeat VI is a high affinity COUP-TF binding site.
  • USF-0 DNA or USF-0 + AVI 12 DNA was cotransfected with a COUP-TF high level expression vector (CMX-COUP-
  • Figure IOC depicts a "designer" adenovirus vector whereby a binding site for the bacterial lac repressor is situated adjacent to and overlapping adenovirus packaging repeat AV.
  • the lac repressor binding site is a perfectly symmetric sequence that binds lac repressor very tightly (Sadler et al . 1983).
  • Lac repressor is a bacterial protein not expressed in eukaryotic cells.
  • Eukaryotic cell, high level expression vectors were generated in our laboratory that express two forms of the lac repressor: 1) the wild type lac repressor, 2) the X86 mutant lac repressor which binds with 40-fold greater affinity to a lac site that the wild type lac repressor.
  • Both forms of lac repressor carry epitope-tag (M45) at the amino-terminus for detection of protein expression in eukaryotic cells by Western blot analysis using a monoclonal antibody against the epitope-tag (mAb M45; Obert et al . 1994).
  • mAb M45 monoclonal antibody against the epitope-tag
  • FIG. 11 shows a Western blot analysis of lac repressor expression in transfected 293 cells showing stable and high level expression of wild type and X86 lac repressors.
  • Fig. 11 also shows a gel mobility shift assay using wild type and X86 Lac repressors expressed in vivo with a DNA probe containing the sequence shown in Fig. IOC. Stable DNA binding to the probe by both repressor forms is evident; specificity for Lac repressor is verified since: a) no binding is evident in cell extracts lacking Lac repressor, and b) the monoclonal antibody against Lac repressor alters the mobility (supershifts) the DNA-protein complex.
  • a recombinant adenovirus was generated that contains two copies of AV-VII + lac (Fig. IOC) in place of the adenovirus type 5 packaging domain (nt 194-814) .
  • CMX + lac viral DNA was cotransfected with the Lac repressor wild type high level expression vector (CMX + lac repressor) or with a control vector (CMX) into human 293 cells. Two days later, production of infectious virus was assayed. The results (Fig. 12) showed that lac repressor expression specifically repressed production of the "designer" virus AV-VII+lac. The maximum level of repression of packaging of AV-VII+lac by lac repressor expression was 20-fold.
  • P complex binds to AT-rich A repeat DNA sequences.
  • An abundant cellular, nuclear protein that binds to such sequences is the TATA binding protein (TBP) which is a cellular transcription factor involved in transcription of cellular promoters.
  • P complex binding is specifically competed using a known, high affinity TBP binding site (TATA box) which is supportive of the idea that P complex may contain TBP.
  • TATA box TATA box
  • P complex also binds to the adenovirus terminus to sequences 1-13. A panel of site-directed points mutations was made through this region to identify the binding site and it was found that all but one of the individual mutations did not reduce P complex binding, while combinations of multiple mutations reduced P complex binding 10-fold or greater.
  • This type of binding pattern is consistent of a protein making interactions with the minor groove of the DNA, instead of the major groove of the DNA. It is known that TBP binds to the minor groove of DNA.
  • TAF172 A protein complex containing TBP plus another protein termed TAF172 has been described (alternatively named TAF170; Timmers et al . 1992, Taggart et al. 1992) .
  • TBP and TAF172/170 are cloned (Hoffman et al . ,
  • TAF172 has intrinsic ATP'ase activity and the
  • TBP-TAF172 complex is displaced from DNA in the presence of ATP + MgCl 2 , as found with P complex and A repeat binding (described above) .
  • a purification scheme, for P complex activity has been developed (Fig. 5) .
  • P complex (identified by mobility shift assay with a DNA probe) and TBP-TAF172 complex (identified by Western blot using anti-TBP and anti-TAF172 antibodies) copurify through each column used with a final P compl3ex purification of ⁇ 1000-fold. Taken together, these results indicate that P complex contains TBP-TAF172.
  • COUP-TF dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitaminD3, thyroid hormone, and retinoic acid receptors. Mol. Cell. Biol. 12: 4153-4163.
  • Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein. Cancer Gene Ther. 3:121- 130. Hirt, B. 1967. Selective extraction of polyoma
  • adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex.
  • the adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex. Mol. Cell. Biol. 14: 1333-1346.
  • helper-dependent adenovirus vector system removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Nat'l Acad. Sci. USA 93: 13565-13570.
  • the upstream factor binding site is not essential for activation from the adenovirus major late promoter. J. Virol. 64: 5851-5860.
  • TATA-binding protein and associated factors are components of pol III transcription factor TFIIIB.
  • COUP transcription factor is a member of the steroid receptor superfamily. Nature 340: 163-166.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Wrapping Of Specific Fragile Articles (AREA)

Abstract

La présente invention concerne des vecteurs d'adénovirus et leur utilisation dans des systèmes d'apport d'ADN. Ces vecteurs ont été conçus pour présenter une capacité améliorée à porter un ADN étranger et un potentiel minimisé de production de virus de réplication. Ces vecteurs contiennent par ailleurs une ou plusieurs copies d'une séquence d'encapsidation minimale, afin de diriger l'encapsidation virale. Ces vecteurs contiennent éventuellement un ou plusieurs sites de liaison de répresseurs, de sorte que la production de virions peut être sélectivement inhibée. L'invention concerne également des systèmes de répression spécifiques, comprenant les répresseurs COUP-TF et lac. L'invention concerne enfin un complexe cellulaire, ci-après dénommé complexe P, fonctionnant de manière positive dans l'encapsidation et la production virales.
PCT/US1999/008294 1998-04-15 1999-04-15 Regulation selective de la production d'adenovirus WO1999053085A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
NZ502175A NZ502175A (en) 1998-04-15 1999-04-15 Selective regulation of adenovirus packaging with COUP-TF or lac repressor
IL13351099A IL133510A0 (en) 1998-04-15 1999-04-15 Selective regulation of adenovirus production
EP99917543A EP0994958A2 (fr) 1998-04-15 1999-04-15 Regulation selective de la production d'adenovirus
AU35636/99A AU770005B2 (en) 1998-04-15 1999-04-15 Selective regulation of adenovirus production
JP55211099A JP2002506355A (ja) 1998-04-15 1999-04-15 アデノウィルス生産の選択的制御

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US8186798P 1998-04-15 1998-04-15
US60/081,867 1998-04-15
US8832198P 1998-06-05 1998-06-05
US60/088,321 1998-06-05

Publications (2)

Publication Number Publication Date
WO1999053085A2 true WO1999053085A2 (fr) 1999-10-21
WO1999053085A3 WO1999053085A3 (fr) 2000-01-27

Family

ID=26766062

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/008294 WO1999053085A2 (fr) 1998-04-15 1999-04-15 Regulation selective de la production d'adenovirus

Country Status (6)

Country Link
EP (1) EP0994958A2 (fr)
JP (1) JP2002506355A (fr)
AU (1) AU770005B2 (fr)
IL (1) IL133510A0 (fr)
NZ (1) NZ502175A (fr)
WO (1) WO1999053085A2 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049166A3 (fr) * 1999-02-18 2001-04-12 Merck & Co Inc Systeme de production de vecteurs d'adenovirus dependant d'auxiliaires base sur l'utilisation d'endonucleases
WO2001053504A1 (fr) * 2000-01-21 2001-07-26 University Of Michigan Medical School Vecteurs adenovirus deficients pour la replication
WO2001040455A3 (fr) * 1999-12-03 2002-01-17 Univ Pennsylvania Compositions et procedes servant a augmenter l'encapsidation et la production d'adenovirus de recombinaison au moyen de signaux multiples d'encapsidation
WO2003027271A3 (fr) * 2001-09-25 2004-03-04 Univ New York State Res Found Regulation de l'encapsidation de l'adn adenovirual par iptg
US6821512B1 (en) 1999-12-03 2004-11-23 The Trustees Of The University Of Pennsylvania Compositions and methods for increasing packaging and yield of recombinant adenoviruses using multiple packaging signals
US6916635B2 (en) 2000-10-02 2005-07-12 The Research Foundation Of State University Of New York Hybrid adenovirus/adeno-associated virus vectors and methods of use thereof
US7135187B2 (en) 1993-06-24 2006-11-14 Advec, Inc. System for production of helper dependent adenovirus vectors based on use of endonucleases
US8309349B2 (en) 2004-10-28 2012-11-13 University of Pittsburgh—of the Commonwealth System of Higher Education Cell line
WO2019020992A1 (fr) * 2017-07-25 2019-01-31 Oxford Genetics Limited Vecteur adénoviral
US12234472B2 (en) 2021-10-18 2025-02-25 Regeneron Pharmaceuticals, Inc. Eukaryotic cells comprising adenovirus-associated virus polynucleotides

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
D'HALLUIN J.C. ET AL.: "Effects of novobiocin on adenovirus DNA synthesis and encapsidation" NUCLEIC ACID RESEARCH, vol. 8, no. 7, 1980, pages 1625-1641, XP002117831 OXFORD, GB *
DULL T. ET AL.: "A third-gneration lentivirus vector with a conditional packaging system" JOURNAL OF VIROLOGY, vol. 72, no. 11, October 1998 (1998-10), pages 8463-8471, XP002117829 BALTIMORE, US *
HUANG M.M., HEARING P.: "The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex" GENES AND DEVELOPMENT, vol. 3, 1989, pages 1699-1710, XP002119862 cited in the application *
O'CONNOR R J ET AL: "THE C-TERMINAL 70 AMINO ACIDS OF THE ADENOVIRUS E4-ORF6/7 PROTEIN ARE ESSENTIAL AND SUFFICIENT FOR E2F COMPLEX FORMATION" NUCLEIC ACIDS RESEARCH, vol. 19, no. 23, 11 December 1991 (1991-12-11), pages 6579-6586, XP000604601 ISSN: 0305-1048 *
SCHMID S I AND HEARING P: "Bipartite structure and functional independence of adenovirus 5 packaging elements" JOURNAL OF VIROLOGY, vol. 71, no. 5, May 1997 (1997-05), pages 3375-3384 3384, XP002108134 ISSN: 0022-538X cited in the application *
SCHMID S.I., HEARING P.: "Cellular components interact with adenovirus type 5 minimal DNA packaging domains" JURNAL OF VIROLOGY, vol. 72, no. 8, August 1998 (1998-08), pages 6339-6347, XP002117828 cited in the application *
SCHMID S.I., HEARING P.: "Selective encapsidation of adenovirus DNA" CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, vol. 199, no. 1, 1995, pages 67-80, XP002117827 *
TAGGART A. ET AL.: "The TATA-bimnding protein and associated factors are components of Pol III transcription factor TFIIIB" CELL, vol. 71, no. 6, 1992, pages 1015-1028, XP002117830 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7135187B2 (en) 1993-06-24 2006-11-14 Advec, Inc. System for production of helper dependent adenovirus vectors based on use of endonucleases
WO2000049166A3 (fr) * 1999-02-18 2001-04-12 Merck & Co Inc Systeme de production de vecteurs d'adenovirus dependant d'auxiliaires base sur l'utilisation d'endonucleases
WO2001040455A3 (fr) * 1999-12-03 2002-01-17 Univ Pennsylvania Compositions et procedes servant a augmenter l'encapsidation et la production d'adenovirus de recombinaison au moyen de signaux multiples d'encapsidation
US6821512B1 (en) 1999-12-03 2004-11-23 The Trustees Of The University Of Pennsylvania Compositions and methods for increasing packaging and yield of recombinant adenoviruses using multiple packaging signals
WO2001053504A1 (fr) * 2000-01-21 2001-07-26 University Of Michigan Medical School Vecteurs adenovirus deficients pour la replication
US6867022B1 (en) 2000-01-21 2005-03-15 Regents Of The University Of Michigan Replication deficient adenovirus vectors and methods of making and using them
US7563617B2 (en) 2000-10-02 2009-07-21 The Research Foundation Of State University Of New York Hybrid adenovirus/adeno-associated virus vectors and methods of use thereof
US6916635B2 (en) 2000-10-02 2005-07-12 The Research Foundation Of State University Of New York Hybrid adenovirus/adeno-associated virus vectors and methods of use thereof
WO2003027271A3 (fr) * 2001-09-25 2004-03-04 Univ New York State Res Found Regulation de l'encapsidation de l'adn adenovirual par iptg
US7585498B2 (en) 2001-09-25 2009-09-08 The Research Foundation Of State University Of New York Regulation of adenovirus DNA packaging by IPTG
US8309349B2 (en) 2004-10-28 2012-11-13 University of Pittsburgh—of the Commonwealth System of Higher Education Cell line
WO2019020992A1 (fr) * 2017-07-25 2019-01-31 Oxford Genetics Limited Vecteur adénoviral
CN110892064A (zh) * 2017-07-25 2020-03-17 牛津遗传学有限公司 腺病毒载体
KR20200031141A (ko) * 2017-07-25 2020-03-23 옥스포드 제네틱스 리미티드 아데노바이러스 벡터
AU2018307569B2 (en) * 2017-07-25 2022-03-10 Oxford Genetics Limited Adenoviral vectors
KR102609021B1 (ko) 2017-07-25 2023-12-06 옥스포드 제네틱스 리미티드 아데노바이러스 벡터
US12071631B2 (en) 2017-07-25 2024-08-27 Oxford Genetics Limited Adenoviral vectors
AU2018307569C1 (en) * 2017-07-25 2025-05-29 Oxford Genetics Limited Adenoviral vectors
US12234472B2 (en) 2021-10-18 2025-02-25 Regeneron Pharmaceuticals, Inc. Eukaryotic cells comprising adenovirus-associated virus polynucleotides

Also Published As

Publication number Publication date
WO1999053085A3 (fr) 2000-01-27
NZ502175A (en) 2002-12-20
AU770005B2 (en) 2004-02-12
JP2002506355A (ja) 2002-02-26
AU3563699A (en) 1999-11-01
EP0994958A2 (fr) 2000-04-26
IL133510A0 (en) 2001-04-30

Similar Documents

Publication Publication Date Title
RU2219241C2 (ru) Дефектный рекомбинантный аденовирусный вектор (варианты)
AU712243B2 (en) Defective adenoviruses including a therapeutic gene and an immunoprotective gene
EP0870049B1 (fr) Systemes de vecteurs adenoviraux et de lignees cellulaires complementaires
KR100403708B1 (ko) 재조합아데노-수반바이러스(aav)제조방법및이의용도
US5994106A (en) Stocks of recombinant, replication-deficient adenovirus free of replication-competent adenovirus
AU708561B2 (en) An adenovirus supervector system
US6482617B2 (en) Viable contaminant particle free adenoviruses, their preparation and use
US20040161848A1 (en) Adenoviral vector and related system and methods of making and use
JP2001527420A (ja) 非ｃ群アデノウイルスベクターの製造方法
WO1995027071A9 (fr) Systeme supervecteur adenoviral
JP2009028040A (ja) 非ｃ群アデノウイルスベクター
CZ230697A3 (en) Cells for producing recombinant adenoviruses
KR19980702139A (ko) 재조합 아데노바이러스 게놈의 제조방법
AU770005B2 (en) Selective regulation of adenovirus production
JP3827163B2 (ja) 遺伝子治療用のアデノウイルスベクター
US20030077828A1 (en) Methods for highly efficient generation of adenoviral vectors
US7264818B2 (en) BAV packaging regions and E1 transcriptional control regions
AU730278B2 (en) Complementary adenoviral vector systems and cell lines
AU703768C (en) Complementary adenoviral vector systems and cell lines
AU2002327630A1 (en) Adenoviral vector and related system and methods of making and use

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 133510

Country of ref document: IL

AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 502175

Country of ref document: NZ

Ref document number: 35636/99

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1999917543

Country of ref document: EP

AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWP Wipo information: published in national office

Ref document number: 1999917543

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWE Wipo information: entry into national phase

Ref document number: 09530935

Country of ref document: US

WWW Wipo information: withdrawn in national office

Ref document number: 1999917543

Country of ref document: EP