JP2002538758A - Method for producing pseudo adenovirus vector - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel helper adenovirus that facilitates the production of a pseudo-adenovirus vector (PAV) but cannot be packaged in virus particles. The invention further relates to a novel PAV producer cell line that expresses a DNA binding protein and / or a repressor protein that prevents packaging of the helper virus. The present invention also relates to a method of producing PAV in such a cell line with minimal contamination with helper virus.

Description

DETAILED DESCRIPTION OF THE INVENTION

Introduction The present invention relates to a novel helper adenovirus that facilitates the production of a pseudo-adenovirus vector (PAV) but cannot be packaged in virus particles.
The invention further relates to a novel PAV producer cell line that expresses a DNA binding protein and / or a repressor protein that prevents packaging of the helper virus. The present invention also relates to a method of producing PAV in such a cell line with minimal contamination with helper virus.

BACKGROUND OF THE INVENTION fake adenoviral vectors (PAV) comprising a minimum cis-acting nucleotide sequence required for packaging of the replication and the vector genome, adenovirus, which may include one or more of the transgenes incorporated Adenovirus vectors derived from the viral genome (see, eg, patent US application Ser. No. 08/89519).
No. 4, incorporated herein by reference). Such PAVs are advantageous because the transgene carrying capacity of the vector is optimized (up to a size of 36 Kb), while at the same time reducing the potential of host immune response to viral proteins or generation of replication competent virus. PAV contains an adenovirus 5 'and 3' inverted terminal repeat (ITR) nucleotide sequence containing an origin of replication, a 5 'cis-acting packaging site of the viral genome, and is capable of activating one or more transgenes Under the control of an expression element linked to These minimal viral nucleotide sequences retained in PAV are required in cis for replication of the PAV genome and packaging into viral particles. Furthermore, the production of PAV requires the provision of a helper virus to supply the viral proteins necessary for replication of the PAV genome and assembly of virus particles. PAV vectors are designed to take advantage of the desirable characteristics of adenovirus, which, as evidenced by studies on first and second generation adenoviruses, make PAV a suitable gene transfer vehicle. . Adenoviruses are non-enveloped, nuclear DNA viruses with a genome of about 36 kb, and are described in classical genetics and molecular biology (Horwitz, MS, "Adenoviruses," in Vir.
ology, 3rd edition, Fields et al., eds., Raven Press, New York, 1996; Hi
tt, MM et al., "Adenovirus Vectors," in The Development of Human Gene
Therapy, Friedman, T. ed., Cold Spring Harbor Laboratory Press, New Yor
k, 1999). Viral genes are divided into early (known as E1-E4) and late (known as L1-L5) transcription units, which give rise to two transient types of viral proteins. These borders are viral DNA replication. Based on properties including hemagglutination, tumorigenicity, DNA base composition and amino acid composition and homology of proteins, and antigenic relevance, human adenoviruses are classified into a number of serotypes (about 47, numbered And organized into six subgroups A, B, C, D, E and F). Recombinant adenovirus has several advantages when used as a gene transfer vector, carrying tropism for dividing and non-dividing cells, minimal pathogenicity, high titer for vector stock preparation, and carries large inserts (Hitt et al., Supra; Berkner, KL, Curr. Top. Micro. Immunol. 1
58: 39-66, 1992; Jolly, D., Cancer Gene Therapy 1: 51-64, 1994). To date, the cloning capacity of adenovirus has been proportional to the size of the adenovirus genome present in the vector. For example, a cloning capacity of about 8 kb would result in the deletion of regions of the viral genes essential for viral growth, such as the E3 region, as well as the ability of E1 to restore its function in trans from a complementary cell line such as 293 cells. (Graham, F. et al.).
L., J. Gen. Virol. 36: 59-72, 1977). Such E1 deletion vectors lack replication, a necessary attribute of gene transfer vectors. The upper limit of the vector DNA carrying capacity for exhibiting the optimum carrying capacity is about 105 to 108% of the length of the wild-type genome. For example, the complement of E2a (Zhou et al., J. Virol. 70: 7030-7038, 1996), E2a
4 (Krougliak et al., Hum. Gene Ther. 6: 1575-1586, 1995; Wang et.
al., Gene Ther. 2: 775-783, 1995), or the complement of protein IX (Caravokyri et al.
al., J. Virol. 69: 6627-6633, 1995; Krougliak et al., Hum. Gene Ther. 6:
Further modification of the adenovirus genome in vector design is possible using cell lines that supply other viral genomic products in trans, such as 1575-1586, 1995). However, maximal carrying capacity can be achieved using adenoviral vectors deleted of most viral coding sequences, including PAV (US Patent Application Ser. No. 08 / 895,194, granted; Kochanek et al., Proc. Natl. A
cad. Sci. USA 93: 5731-5736, 1996; Parks et al., Proc. Natl. Acad. Sci.
USA 93: 13565-13570, 1996; Lieber et al., J. Viol. 70: 8944-8960, 1996;
Fisher et al., Virology 217: 11-22, 1996; U.S. Patent No. 5,670,488;
PCT publication WO 96/33280 published October 24, 996; December 1996
PAC publication WO96 / 40955 published on March 19; PAC publication WO97 / 25446 published on July 19, 1997; PCT publication WO published on November 9, 1995
95/29993; PCT publication WO 96/13597 published on May 9, 1996.
PCT Publication WO / 00326 published Jan. 3, 1997; Marral et al /., Hu
m. Gene Ther. 10: 2709-2716, 1988; Burcin et al., Proc. Natl. Acad. Sci.
USA 95: 355-360, 1999). A wide range of transgenes (foreign nucleic acids) have been delivered to a variety of target cells using first and second generation adenovirus vectors, explaining the heterogeneity of transduction with adenovirus vectors. Such a transgene is p53
(Mills et al., Human Gene Therapy 5: 1079-188, 1994), dystrophin (
Vincent et al., Nature Genetics 5: 130-134, 1993), erythropoietin (
Descamps et al., Human gene Therapy 5: 979-985, 1994), ornithine transcarbamylase (Stratford-Perricoudet et al., Human Gene Therapy 1:24).
1-134, 1993; We et al., J. Biol. Chem. 271: 3639-3646, 1996), adenosine deaminase (Mitani et al., Human Gene Therapy 5: 941-948, 1994), interleukin-2 (Haddada et al., Human gene Therapy 4: 703-711, 1993)
And α1-antitrypsin (Jaffe et al., Nature genetics 1: 372-3
78, 1992), Thrombopoietin (Ohwada et al., Blood 88: 778-784, 1996)
And cytosine deaminase (Ohwada et al., Hum. Gene Ther. 7: 1567-1
576, 1996). The particular tropism of adenovirus for respiratory tract cells has been associated with the use of adenovirus for gene transfer in cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which disrupts cAMP-regulated Cl-channels in airway epithelium, cause lung dysfunction (Zabner e
tal., Nature Genetics 6: 75-83, 1994). Adenovirus vectors that have been engineered to carry the CFTR gene have been developed (Rich et al., Human Gene Ther.
apy 4: 461-476, 1993), studies have shown that nasal epithelium in CF patients (Zabner et al., Cel)
l 75: 207-216, 1993), respiratory epithelium of cotton rats and primates (Zabner et a
l., Nature Genetics 6: 75-83, 1994), and respiratory epithelium of CF patients (Crys
tal et al., Nature Genetics 8: 42-51, 1994) show the ability to deliver CFTR. Recent studies have shown that administration of adenoviral vectors containing DNA sequences encoding CFTR to airway epithelial cells of CF patients can restore chloride ion channel function in treated epithelial cells (Zabner et al. al
., J. Clin. Invest. 97: 1504-1511, 1996; U.S. Patent No. 5,670,488, issued September 23, 1997). Sustained CFTR expression from adenoviruses that establish functional chloride channels in the respiratory epithelium of immunocompromised animals has recently been achieved (Scaria et al., J. Virol. 72: 7302-7309, 1998). At present, the use of first and second generation adenovirus vectors in gene transfer studies has shown that the maintenance of the transgene in target cells and tissues is often transient. This means that, at least in part, even viral proteins expressed from replication-defective vectors can elicit a cellular immune response, destroy adenovirus-infected cells or adversely affect the pathological inflammatory response. Because it triggers (Yang et al., J. Virol. 69: 2004-2015, 1995; Ya
Natl. Acad. Sci. USA 91: 4407-4411, 1994; Zsengeller et.
al., Hum. Gene Ther. 6: 457-467, 1995; Worgall et al., Hum. Gene Ther.
8: 37-44, 1997; Kaplan et al., Hum. Gene Ther. 8: 45-56, 1997). Since adenovirus is not integrated into the cell genome, adverse immune responses impair high or repeated administration of adenovirus (Crystal, R., Science
270: 404-410, 1995). Various strategies have been used to overcome host immune responses that limit the maintenance of transgene expression, and generally include the modulation of the immune response itself or the production of vectors that reduce the immune response. Administration of immunosuppressants with vector administration has been shown to extend transgene maintenance time (Fang et al., Hum. Gene
Ther. 6: 1039-1044, 1995; Kay et al., Nature Genetics 11: 191-197, 1995.
; Zsellenger et al., Hum.Gene Ther. 6: 457-467, 1995; Scaria et al., Ge
ne Therapy 4: 611-617, 1997). Modification of the adenovirus genome in a recombinant vector can reduce the host immune response (Yang et al., Nature Geneti
cs 7: 362-369, 1994; Lieber et al., J. Virol. 70: 8944-8960, 1996; Gorzi
glia et al., J. Virol. 70: 4173-4178; Kochanek et al., Proc. Natl. Acad.
Sci. USA 93: 5731-5736, 1996; Fisher et al., Virology 217: 11-22, 1996.
). For example, the adenovirus E3 gp19K protein forms a complex with MHC class I antigens and retains them in the endoplasmic reticulum, which prevents cell surface presentation and killing of infected cells by cytotoxic T-lymphocytes (CTL). (Wold et al., Trends
Microbiol. 437-443, 1994), E3 gp in a recombinant adenovirus vector.
It is suggested that the presence of the gene encoding the 19K protein is beneficial. The lack of sustained expression of the transgene delivered by the adenovirus vector may be due to limitations due to the choice of the transgene contained in the promoter and / or transcription unit (Guo et al., Gene Therapy 3: 802). -801, 1
996; Tripathy et al., Nature Med. 2: 545-550, 1996). For further optimization of minimal adenoviral vectors for sustained transgene expression in target cells, select expression-control elements and the vector genome to be synergistic to maximize expression and maximize host immune response Need to be restricted (WO98
/ 46780; Scaria et al., J. Virol. 72: 7302-7309, 1998).

Although it is not possible to elicit a strong host immune response, it is desirable to provide PAVs with a minimal viral coding sequence that can harness the capabilities of adenoviral vectors to deliver the transgene to a wide range of target cells. The production of PAV requires the presence of adenoviral proteins that facilitate replication of the PAV genome and packaging into the viral genome. Most commonly, such proteins are provided by infecting producer cells with a helper adenovirus containing a gene encoding such a protein. However, if such helper virus can replicate and be packaged in viral particles, such helper virus is potentially a source of contamination of the PAV stock during purification. It is therefore desirable to facilitate the purification of PAV stocks by reducing or eliminating the production of helper virus that can contaminate the preparation. Several strategies for reducing the production of helper virus in PAV stock formulations are disclosed in US patent application Ser. No. 08 / 895,194 and US Pat. No. 5,670,488, issued Sep. 23, 1997, which is incorporated herein by reference. ). For example, the helper virus may have packaging disruption mutations in the packaging sequence in its genome, or may contain an oversized adenovirus genome that cannot be packaged due to size limitations of the viral particles. Good. Another strategy involves designing the helper virus to flank the packaging signal by excision of the recombinase target site, such as the cre-lox system (Parks et al., Peoc. Natl. Acad. . Sci. USA 93: 13565
-13570, 1996; Hardy et al., J. Virol. 71: 1842-1849, 1997). Detailed analysis of the adenovirus packaging signal revealed that it was organized into a minimum of seven elements identified as A repeats (Schmid et al., J. Virol. 71: 3375- 3384, 1997). Using this information, PA
A PAV generation system is provided that includes a helper adenovirus and a producer cell line optimized for V production, such that the packaging signal region is available for helper virus stock production, but is disabled during production of the PAV vector stock. Be transformed into The present invention facilitates the preferred packaging of PAVs in the production of purified vector stocks for further development and widespread use of these vectors for gene transfer.

SUMMARY OF THE INVENTION The present invention relates to a novel helper adenovirus, which provides in trans the production of essential viral proteins required for the production and packaging of a pseudo-adenovirus vector (PAV). By doing, PA
Facilitates V generation and packaging. The helper virus of the present invention contains a binding site for a DNA binding protein and / or a repressor protein (which prevents the packaging protein from accessing this signal) in the packaging signal region of its genome, Packaging is missing. The present invention also provides
It relates to a PAV producer cell line that expresses a nucleic acid encoding such a DNA binding protein and / or a repressor protein. The present invention further relates to a method for producing PAV using the helper virus and the producer cell line of the present invention, which minimizes the contamination of PAV with helper virus.

[0005] DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel helper adenovirus, the helper adenovirus to generate essential viral proteins necessary for the formation and packaging of the fake adenoviral vectors (PAV) trans state By providing at the PA
Facilitates V generation and packaging. The helper virus of the present invention contains a DNA binding protein and / or a repressor protein in the packaging signal region of its genome (which prevents access by the packaging protein to this signal or enzymatic excision of the packaging signal). Packaging site since the binding site is included. The invention also relates to PAV producer cell lines expressing nucleic acids encoding such DNA binding proteins and / or repressor proteins. The present invention further relates to a method for producing PAV using the helper virus and the producer cell line of the present invention, which minimizes the contamination of PAV with helper virus.

[0006] A helper virus of the invention is defined as an adenovirus capable of supplying the viral proteins required in trans for the production of PAV or other minimal adenoviral vectors. According to the present invention, the helper virus genome is not packaging capable, thus allowing preferential packaging of the PAV genome into virus particles. The helper virus genome contains at least the genes and / or regions of the adenovirus genome required to make the viral proteins required in trans for the production of PAV. Adenovirus proteins provided by the helper virus include, inter alia, regulatory proteins from the adenovirus early (E) genomic region, capsid proteins encoded by the viral late (L) genomic region, and other structural and nonstructural proteins. I do. Generation of the protein encoded by the helper virus genome facilitates replication of the PAV genome during vector stock production. The adenoviral genes required in trans are not limited by the viral serotype, and the helper virus of the invention may include adenoviral genes derived from more than one serotype. Therefore, the structural proteins provided by the helper virus can be selected such that the capsid protein is derived from the desired serotype (s) and is optimized for a particular application. Desirably, the helper virus genome is modified according to the invention to reduce or eliminate helper-virus particle packaging. Such disabling reduces or eliminates the production of helper virus during PAV stock preparation, while allowing the helper virus itself to grow while making the helper virus stock separately. Incorporating a reduced-length packaging signal into the genome of the helper virus of the present invention, or inserting a heterologous nucleotide sequence (defined as a binding sequence) in or near the packaging signal region in the helper virus genome This renders the helper virus of the present invention defective in packaging. Such a binding sequence is specific DNA
Can bind to a binding protein and / or a repressor protein, thus blocking the use of a cis-acting packaging signal (masking the packaging signal) or causing excision of the signal from the helper virus genome (package Of the signaling signal) (FIG. 1).

[0007] As used herein, a binding protein is a protein or peptide, which can (1) bind to a binding sequence inserted into or near the packaging signal region of the helper virus genome, or (2) specific target It is defined as capable of binding to a site and inducing cleavage. A binding sequence is defined as a nucleotide sequence inserted near, adjacent to, or within a helper virus genome, which can bind to a specific DNA binding protein and / or repressor protein. The binding sequences of the present invention can be used alone or in combination with other binding sequences or in parallel with them.
One or more of the DNA binding proteins and / or repressor proteins, so that the viral packaging protein cannot be in close proximity to the packaging signal of the helper virus genome, or Signaling is excised from the genome. Preferably, the binding sequence interacts with and binds a DNA binding protein and / or a repressor protein. The binding sequence incorporated into or near the packaging signal region of the helper virus genome may be of various lengths, for example, about 8-30 nucleotides, but such sequences may be juxtaposed. Preferred specific binding sequences of the present invention are derived from bacteriophage lambda operators (Ptashne, M. A Genetic Switch, Cell Press and Blackwell Scientific
Publications, 1986) and from the papillomavirus E2 binding sequence (McBride et al.
, J. Biol. Chem. 266: 18411-18414, 1991). Most preferably, the packaging signal region in the helper virus FLP recombinase, which recognizes the FLP recombinant target (FRT) and can catalyze the catalytic site-specific excision of flanking nucleotide sequences (Senecoff et al., Proc. Natl.
Acad. Sci. 82: 7270-7274, 1985; O'Gorman et al., Science 251: 1351-1355, 1991). FLP by FLP recombinase
If the RT nucleotide sequence is recognized, the flanking packaging signal is excised from the helper virus genome, which prevents packaging of the helper virus genome and production of helper virus particles (FIG. 2). In a preferred embodiment of the invention, FLP recombinase is used in the producer cells of the invention to enzymatically cleave the packaging signal in the helper virus. Because it shows improved thermal stability at 37 ° C. (Buch
holz et al., NAR 24: 4256-4262, 1996; Buchholz et al., Nature Biotech. 1
6: 657-662, 1998). The present invention contemplates the use of FLP recombinases, such as monomers, dimers, tetramers, or other multimers, that are optimized for the particular application required.

[0008] Specific binding sequences that can be inserted into the adenovirus packaging signal region of the adenovirus genome at or near the site to achieve the objects of the present invention include: Lambda operator binding sequences (Ptashne, M. A Genetic Switch, Cell Press a
nd Blackwell Scientific Publications, 1986): O L 1: TATCACCGCCAGTGGTA ( SEQ ID _{NO: 1) ATAGTGGCGGTCACCAT O R 1:} TATCACCGCCAGAGGTA ( SEQ ID _{NO: 2) ATAGTGGCGGTCTCCAT O L 2:} TATCTCTGGCGGTGTTG ( SEQ ID _{NO: 3) ATAGAGACCGCCACAAC O L 3:} TATCACCGCAGATGGTT ( SEQ ID _{NO: 4) ATAGTGGCGTCTACCAA O R 2:} TAACACCGTGCGTGTTG ( SEQ ID _{NO: 5) ATTGTGGCACGCACAAC O R 3:} TATCACCGCAAGGGATA ( SEQ ID NO: in 6) ATAGTGGCGTTCCCTAT preferred embodiment, binding sequences O _L 1: TATCACCGCCAGTGGTA (SEQ ID NO: 1) ATAGTGGCGGTCACCAT It is. Specific binding sequences used by papillomavirus E2 protein include: ACCGAAATCGGT (SEQ ID NO: 7) Romanczuk et al., J. Virol. 64: 2489-2859, 1990 ACCGAAACCGGT (SEQ ID NO: 8) Romanczuk et al., J. Virol. 64: 2489-2859, 1990 ACCN (6) GGT (SEQ ID NO: 9) Androphy et al., Nature 325: 70-73, 1985 Others that can be used as binding sequences of the invention The papillomavirus E2 protein consensus sequence is that described by McBride et al., J. Biol. Chem. 266: 18411-18414, 1991. The binding sequence that responds to FLP recombinase is based on its recognition site, the FLP recombinase target (FRT) repeat motif (Senecoff et al., Proc. Natl. Acad. Sci.
USA 82: 7370-7274, 1985). FRT: GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC (SEQ ID NO: 10) Another binding site for FLP recombinase is: CGAAGTTCCTATTCTCTAGAAAGtATAGGAACTTC (SEQ ID NO: 11). The FRT binding sequence may be derived from plasmid pOG45 (Stratagene, La Jolla, CA) or may be synthesized using standard methods for oligonucleotide synthesis. Truncated FRT sequences that facilitate the cleavage of adjacent packaging signals in the helper virus are also within the scope of the invention. FLP
Use of a binding sequence to facilitate the excision of a packaging signal from a helper adenovirus mediated by is within the scope of the present invention in the construction of a packaging defective helper adenovirus.

The nomenclature of the packaging signal region (referred to as AI-AVII) that defines the insertion position of the binding sequence in or near the packaging signal in the adenovirus genomic region, defining the smallest of the seven AT-rich elements Can be confirmed by reference. These seven AT-rich elements are present at nucleotides 194-380 of the adenovirus genome (see adenovirus serotype 5, Schmid e).
t al., J. Virol. 71: 3375-3384, 1997). For example, one or more binding sequences can be inserted at one or more A element flanking sites or between A repeat elements. Redesign of the adenoviral packaging signal region according to the invention also contemplates deletion or multiplexing of one or more A-repeat elements. Methods for designing adenovirus will be apparent to those skilled in the art, and the redesign may involve various combinations of insertions of the binding sequence, or insertion of the binding sequence at or near the A repeat element, and deletion or repetition thereof. Embrace,
It achieves the object of the present invention, that is, it neutralizes the packaging signal of the helper virus genome. If the binding sequences facilitate excision of the packaging signal, they may be inserted into the adenovirus genome and flank the 5 'and 3' ends of the desired packaging signal region, so that the entire signal region May be excised by a site-specific recombinase.

In one preferred embodiment of the helper virus packaging signal,
The Luper virus genome is repaired by the deletion of the packaging elements AI to AIV.
Decorated, only the packaging elements AV, AVI and AVI are retained
It is. A 17 base pair bacteriophage lambda operator sequence (preferably O _L A binding sequence comprising 1) upstream of element AV in the helper genome and AVI
Downstream of I (sites 1 and 2), ie adjacent to nucleotides 334 and 385
(FIG. 3: Ad (AV-AVI-AVII). Alternatively, FIG.
As shown, the repetition using the repetitive elements AV, AVI and AVII as motifs
The inserted lambda operator, for example, as (AV-AVI-AVII) 2
(FIG. 4: Ad (AV-AVI-AVII) 2)
). Helper vectors Ad (AV-AVI-AVII) and Ad (AV-AV
I-AVII) 2 can be used directly as a helper [Ad (AV-AV
In the case of I-AVII), packaging is deficient] or the protein encodes
Can be used with lambda repressors in nucleic acid expressing cell lines
(Helper vector with packaging masked).

In another preferred embodiment, the packaging signal region of the helper virus genome comprises a packaging element AI-AII-AIII-AIV
And the packaging elements AV, AVI and AVII
Only to be kept. Operator sequence 17 base pairs from bacteriophage lambda (preferably, O _L 1) between the elements AV and AVI of a helper virus genome binding sequence comprising (part 3), and AV upstream and AV
Insert downstream of II (sites 1 and 2) (FIG. 5). In a further preferred embodiment, the packaging signal region of the helper virus genome is modified by deleting the packaging elements AVI and AVII. Operator sequence 17 base pairs from bacteriophage lambda (preferably, O _L 1) a binding sequence containing in the helper virus genome A
Insert between II and AIII (site 5) and upstream of AI and downstream of AV (sites 4 and 6) (FIG. 6).

[0012] The binding sequence (IV) in the packaging signal region of the helper virus genome
Preferred sites for insertion of the II repeat numbered A) include, but are not limited to, the following: Table 1 Sites Position 1 for the A repeat 5'2 AVII for position 1 AV In another preferred embodiment of the 3 ′ helper virus between 3 ′ 3 AV and AVI 5 ′ 5 AII and AIII for 4 AI and 3 ′ for AV In papilloma virus E2
A binding sequence that binds to the protein is inserted into the helper virus genome so as to be adjacent to all or part of the packaging signal region so that the E2 protein is bound. The insertion sites shown in Table 1 are preferred. If the binding sequence includes a lambda operator sequence used with the lambda repressor to prevent packaging of the helper virus during PAV production, it is preferred that the packaging signal region be adjacent to the operator sequence. This is because the cooperative binding of the tetramer to the pair of operator sites is required for complete repression (Ptashne, M. A Genetic Switch, Cell P
ress and Blackwell Scientific Publications, 1986). Previous studies have shown that the cis-acting signal can be blocked using a lambda operator / repressor system, such as the TATA transcription element (Wedler et al., Mol. Gen. Gen.
et. 248: 499-505, 1995).

Where the binding site comprises a FRT binding sequence, particular designs of the packaging signal region of the helper virus genome include, but are not limited to:
(See A-repeat elements numbered I-VII) (FIG. 7): FRT (V-VI-VII) FRT FRT (V-VI-VII) ₂ FRT FRT (V-VI-VII) ₃ FRT FRT (VI ) ₁₂ FRT FRT (VI) ₁₂ lucFRT Packaging signal region FRT (VI) 12FRT or FRT (V-V
I-VII) A preferred embodiment of a helper adenovirus comprising 2FRT is Ad2
HelpFRT is included (FIG. 8). Other combinations of deletions and insertions in or near the packaging signal region of the helper virus genome are within the scope of the invention and create a helper virus incapable of packaging PAV in the cell lines of the invention. Will be recognized by those skilled in the art. If the binding sequence is indicated by (X) and the A-repeat element is indicated by Roman numerals I through VII and the subscript indicates the number of repeats of such motif, other recombinant packaging signal regions include: : XVXVIXVII XIXIIXIIIXIVXVXVIXVIIX X (I-II-III-IV-V-VI-VII) XX (I-II-III-IV-V-VI-VII) ₂ XX (I-II-III-IV-V-VI- VI) ₃ XX (I-II-III-IV-V) XX (I-II-III-IV-V) ₂ XX (I-II-III-IV-V) ₃ XX (V-VI-VII) XX (V-VI-VII) ₂ XX (V-VI-VII) ₃ XX (VI) ₁₂ XX [(VI) ₁₂ ] ₂ XX [(VI) ₁₂ ] ₃ X

[0014] Another aspect of the present invention relates to the use of adenovirus genomic sequences in a packaging cell line between a helper virus packaging signal region and a PAV, or a packaging signal region or other region in the helper virus genome (eg, For example, the E1 sequence (nucleotides 1 to 5 of adenovirus serotype 5) in 293 cells
4344)), which relates to a helper virus designed to reduce or prevent recombination and prevent the outbreak of live helper adenovirus due to the recombination. A replication-defective adenovirus vector that leads to the generation of a replicable adenovirus and 2
Recombination with 93 cells was demonstrated (Hehir et al., J. Virol. 70: 8459-846).
7, 1996). If the helper virus genome contains a binding sequence adjacent to the packaging signal region (eg, cleaved by the cre enzyme provided by the packaging cell line (Parks et al., Proc. Natl. Acad. Sci. USA 93: 13565). -135
70, 1996), or a lox site containing the FRT target site of the FLP recombinase), a recombination between the homologous nucleotide sequence in the helper virus genome and the producer cell may result in the deletion of an adjacent target sequence, eg, a lox site or a FRT site. Could lead to Therefore, such a deletion would prevent the excision of the packaging signal in the helper virus mediated by FLP or cre, and reduce the packaging of the helper virus genome during PAV production and packaging. Accordingly, the present invention also provides a helper adenovirus comprising a packaging signal region that is not homologous to the PAV genome. In a particular embodiment of this aspect of the invention, one or more AVI packaging signal sequences that have minimal overlap with any nucleotide sequence in the PAV vector (see Signal Elements, Schmid et al., J. Virol. 71: 3375-3384, 1997) (e.g., packaging signal elements AI-AV).
US Patent No. 08 / 895,194, which contains only nucleotides 1-356 containing
And U.S. Patent No. 5,670,488). Therefore, the recombination potential between PAV and the helper virus genome is significantly reduced.

[0015] In a particularly preferred embodiment of the helper virus genome, the signal region comprises a single (12xAVI repeat) nucleotide sequence or comprises a parallel repeat of this signal. Such a helper virus does not contain a sequence overlapping with the PAV genome, but uses a packaging signal containing a nucleotide sequence derived from A repeat elements IV. Such a helper packaging signal region may be a binding sequence that facilitates excision of the packaging signal by a recombinase (eg, lox, FRT) or binding to a packaging signal by a repressor protein of the invention (eg, a lambda repressor). May be adjacent to a binding sequence that facilitates Insert the marker gene into the helper virus packaging signal region,
Marker genes such as luciferase (assay kits are from Promega, Madison
, Commercially available from WI) due to the loss of the characteristic signal
It may act as a marker for excision of the packaging signal. The preferred genome of the marker helper adenovirus is Ad2HelpFRTluc (
FIG. 9), which shows that once the packaging signal adjacent to the FRT is excised by the FLP recombinase, the signal from the luciferase protein can no longer be detected. However, recombination with a homologous nucleotide sequence in the producer cell line may result in loss of the marker signal, even though the packaging signal has not been deleted. Therefore, in producer cell lines that do not contain a nucleotide sequence that could result in recombination with the 5 'region in the helper virus and could cause misinterpretation of the marker assay for excision of the packaging signal by the FLP recombinase. The marker helper adenovirus is optimally used. For example, PER
. C6 (including nucleotides 459 to 3510 of adenovirus serotype 5)
(The genome contains an adenovirus with minimal overlap with the 5 'nucleotide sequence in the helper virus, thereby reducing recombination potential (Fallaux et al., Hum. Gene. Ther. 1: 1909-1917, 1998) may use the marker helper adenovirus. Preferably, PER
. C6 cells are used in the methods of the invention to minimize recombination between producer cells and helper adenovirus.

In a further embodiment of the invention, a helper having reduced recombination potential between the helper virus genome and a complementary cell line or overlapping adenovirus nucleotide sequence in PAV (eg, the E1 sequence in 293 cells). A virus is provided (such recombination results in a live helper virus contaminating the PAV preparation). When recombination occurs between the helper virus and either the packaging cell or the PAV genome, the helper virus genome is present in the reverse orientation so that the resulting recombinant product does not contain live adenovirus. Contain packaging signals. The packaging signal of such a helper virus is that of an A repeat element (Schmid et al., J. Virol. 71: 3375-3384, 1997) inserted in the genome in the reverse orientation, sufficient to confer packaging ability. Including any combination. In a particularly preferred embodiment, the packaging signal region contains one inverted (12xAVI repeat) sequence.

In a further embodiment of the present invention there is provided a helper virus which is inactivated by recombination with a complementary cell line, preferably an E1 complementary cell line, eg 293 cells, to obtain a homologous sequence. For example, the helper virus is designed so that when recombination occurs, the virus genome becomes too large to be packaged. A preferred embodiment is that the genome contains a packaging signal adjacent to the FRT and that the E3 region of the adenovirus genome in the genome is 2.9 kb.
Is a helper virus TBTP in which a 1.8 kb EGFP gene is operably linked to the CMV promoter and a 5.0 kb fragment of the human alpha-antitrypsin gene is inserted (FIG. 10). ). Although the size of the helper virus is 36.9 kb (102% of wild type), recombination with the adenovirus E1 sequence in 293 cells results in a size of the helper virus genome of about 39.6 kb (110%). Too can not be packaged.

To produce a special helper virus of the present invention, a wild-type or recombinant adenovirus genome can be obtained by inserting a binding sequence into or adjacent to the A-repeat element of the packaging signal region. To modify DN
The A binding protein and / or the repressor protein prevents access to the cis-acting packaging signal when bound to the specific binding sequence. Restriction enzyme digestion and ligation,
Recombinant signal regions can be created in the helper adenovirus genome using standard methods of molecular biology, such as the polymerase chain reaction as well as site-directed mutagenesis. The packaging signal region can then be inserted into a suitable site in the 5 'region of the adenovirus genome using a plasmid containing the signal. Such a plasmid is co-transfected into a cell line with DNA encoding the remainder of the adenovirus helper genome to be included in the helper virus to cause homologous recombination, thereby providing the desired recombinant packaging signal. An associated helper adenovirus can be obtained. Helper virus genomes can be constructed in bacteria, thus simplifying the procedure (Chartier et al., J. Virol. 70:
4805-4810, 1998). Transfection of a plasmid from which non-viral (ie, bacterial) sequences have been removed can provide the entire viral genome. Other methods for producing recombinant adenovirus are known to those of skill in the art, and such methods can be used to obtain a helper virus of the invention.

The helper virus of the invention is derived from a wild-type adenovirus, a truncated or mutated adenovirus, the genome of which encodes the viral proteins required in trans to produce PAV upon introduction. . Furthermore, the helper virus of the present invention is not limited by serotype. Preferably, as a further safety feature for use in the production of PAV for use in gene transfer, the helper virus of the invention is also replication defective. For example, a replication-defective virus can be created by deleting the E1 region of the adenovirus genome. Such helper viruses can grow in E1 complementing cell lines, such as the 293 cell line (Graham et al., J. Gen. Virol. 36: 59-72, 1977).

Although the present invention provides packaging-deficient helper adenoviruses, preferably, the genome of such viruses is optimized for replication and gene expression to obtain sufficient levels of adenovirus helper virus proteins. Therefore, in one particular embodiment of the present invention, the helper virus genome comprises an internal ITR sequence located downstream of the blocked packaging signal region, wherein the internal ITR sequence comprises:
It allows sufficient replication and expression of the helper virus genome in PAV producer cells, thereby ensuring sufficient provision of the required viral proteins in trans. Although the repressor protein occupies a binding sequence inserted in or near the helper virus packaging signal, this particular embodiment of the present invention provides for the replication of the helper virus genome, the adenovirus DN
Provides the exposed ITRs utilized by A polymerase and terminal proteins (FIG. 11).

In a further aspect of the invention, there is provided a method of producing a PAV vector using a PAV genome and a helper adenovirus, wherein the helper adenovirus comprises a packaging signal region or ITR sequence from a different adenovirus serotype, Sequence duplication between the genome and the helper adenovirus is minimized and recombination potential is reduced.

The invention further relates to a PAV producer cell line that produces a DNA binding protein and / or a repressor protein that can bind to a binding sequence inserted in or near the packaging signal region of the helper virus genome. As a result of this interaction, the helper virus is disabled or deleted for packaging during PAV stock production. In a preferred embodiment of the invention, the 293 cells contain and express a nucleic acid encoding a DNA binding protein and / or a repressor protein, thereby creating a PAV producer cell line of the invention.
Other cell lines can be used, including VK2-20 and adenovirus genomic region E2a (Zhou et al., J. Virol. 70: 7030-7038, 1996), E4 (Krougliak).
et al., Hum. Gene Ther. 6: 1575-1586, 1995; Wang et al., Gene Ther. 2: 7
75-783, 1995) or protein IX (Caravokyri et al., J. Virol. 69: 6627-6633).
, 1995; Krougliak et al., Hum. Gene. Ther. 6: 1575-1586, 1995), including but not limited to cell lines designed to complement the deletion. Preferably, the 293 cells contain a DNA binding protein, preferably a tetracycline gene control system (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551,
1992; and Gossen et al., Science 268: 1766-1769, 1995, incorporated herein by reference) and expresses a nucleic acid encoding FLP or a FLP recombinase. In this system, gene expression from the minimal promoter is under strict control of the tetracycline repressor (TetR), and the expression product is a fusion protein (T
etR / VP16). Two versions of TetR / VP16
Protein is present. Wild-type TetR is only active in the absence of tetracycline or doxycycline, and mutant forms of TetR (reverse TetR or rTetR)
Is active only in the presence of doxycycline. When connected to VP16,
TetR activates transcription only in the absence of tetracycline, and rTetR activates transcription only in the presence of doxycycline. These transcription factors Tet
R / VP16 and rTetR / VP16 each regulate the expression of genes linked to a minimal promoter integrated adjacent to a tetracycline transcriptional regulatory element (TRE), such as the FLP recombinase gene, which is stably integrated in 293 cells. I do. A 293 cell line containing a nucleic acid encoding a Tet-off fusion protein (TetR-VP16) is generated as described by the manufacturer (Clontech, Palo Alto, CA) or by transfection with a Tet-off plasmid available from Clontech. be able to. In addition, 29 expressing FLP recombinase
Using a 3-Tet-off cell line, the helper adenovirus packaging signal region flanked by FTR binding sequences can be induced and preferentially excised, or the desired adenovirus genome flanked by the required FRT binding sequences is desired. It is noted that segments can be ablated.

In this embodiment of the invention, the nucleic acid encoding the FLP recombinase is pT
The FLM recombinase was operably linked to a minimal CMV promoter and TetR-integrated into a RE plasmid (Clontech, Palo Alto, CA).
Being under the control of seven tet operator sites responsive to the VP16 fusion protein (FIG. 13a). No transcription from the FLP recombinase gene occurs when tetracycline is provided to 293Tet-off-producer cells (FIG. 13b). Modulation on the expression of FLP recombinase in a dose-dependent manner, and therefore on excision of the helper adenovirus packaging signal catalyzed by FLP, may occur in proportion to the level of tetracycline provided to the producer cells. is there. Preferably, the producer cell line of the invention is a 293 cell line comprising a Tet-off fusion protein under control of a TRE operator sequence and a minimal CMV promoter and a nucleic acid encoding a FLP recombinase, wherein the PAV genome and FRT of the invention are The cell line modulates the production of FLP recombinase when a helper virus is provided that contains a packaging signal adjacent to the binding sequence, thereby preferentially disabling the helper virus for paging during PAV vector production. . Producer cell lines that stably express a nucleic acid encoding FLP recombinase can also be constructed using a plasmid containing a gene encoding FLP recombinase under the control of a suitable promoter, such as CMV or SV40. An example of such a plasmid containing the FLP recombinase gene is pSVK / FL
Pe6 (including FLP recombinase gene under control of SV40 promoter)
(FIG. 14) and pCEP / FLP36 (including the FLP recombinase gene operably linked to the CMV promoter) (FIG. 15). If it is desired to provide the FLP recombinase gene to the cells for rapid expression and perform the assay, a plasmid maintained extrachromosomally, such as pCEP / FLPe6 (FIG. 1)
5) can be used.

The nucleic acid encoding the DNA binding protein and / or the repressor protein can be stably expressed or inducibly expressed by treating the nucleic acid by a standard method of molecular biology into a PAV producer cell. Preferred DNA binding proteins and / or repressor proteins used in the present invention are bacteriophage lambda repressor (wild-type and / or N-terminal fragment), Cro protein (Ptas
hne, MA Genetic Switch, Cell Press and Blackwell Scientific Publication
s, 1986), bovine papilloma E2 protein (McBride et al., J. Biol. Chem. 266: 18411).
-18414, 1991), tetracycline repressor (Gossen et al., Proc. Natl.
Acad. Sci. USA 89: 5547-5551, 1992), tryptophan repressors, as well as other proteins known to those of skill in the art. A specific recombinase of the invention that can be used to excise the packaging signal adjacent to the appropriate binding sequence is FLP recombinase (Broach et al., Cel.
l 21: 501, 1980) or FLPe recombinase (Buchholz et al., NAR 24:
4256-4262, 1996). Other DNA binding proteins and / or repressor proteins that can bind to the relevant binding sequences in the helper virus genome can be used in the cell lines of the invention. For example, a fusion protein of such a repressor protein can also be constructed by creating a nucleic acid encoding β-galactosidase of E. coli linked to a ramuda repressor protein, whereby such a fusion protein can be constructed. When binding to a binding sequence inserted in or near the packaging signal in the helper virus genome, it provides a robust physical obstacle to packaging of the helper virus genome. Activator protein (see tet repressor / HSV VP16; Gossen et al., Proc. Natl.
Acad. Sci. USA 89: 5547-5551, 1992). A fusion protein can also be prepared by preparing a hybrid nucleic acid encoding a DNA binding protein and / or a repressor protein.

The lambda repressor protein monomer is 236 amino acids long (26 KD) and the repressor protein dimer binds to a single 17 bp operator sequence.
Six lambda operator sites with the central base as the axis of symmetry are recognized by the lambda repressor protein dimer in order of their unique affinity for the lambda repressor dimer. The N-terminal protein domain of the lambda repressor recognizes the operator and can be used for binding. In one embodiment of the invention, the C-terminal domain of the repressor can be replaced, for example, by a dimeric form of the leucine zipper protein. To create a PAV producer cell line of the present invention, a nucleic acid encoding a DNA binding protein and / or a repressor protein and the nucleotide sequence of an operably linked regulatory element are introduced into the cell line by any nucleic acid transfer method. To be introduced. Nucleic acid transfer methods include, but are not limited to, transfection, electroporation, or virus-mediated transfer. A plasmid containing a nucleic acid encoding a DNA binding protein and / or a repressor protein and a nucleotide sequence corresponding to a regulatory element can be transfected into the target cell. Where the plasmid further comprises a nucleic acid encoding a selectable marker, such a marker can be used to detect incorporation of foreign plasmid DNA. For example, a nucleic acid encoding neomycin resistance can be introduced alongside a nucleic acid encoding a repressor protein, whereby stably transfected cells can be selected by culturing in the presence of G418. Alternatively, a nucleic acid encoding such a repressor protein can be transferred to an extrachromosomal plasmid maintained as an episome (eg, pCEP-4 (Invitrogen, San Diego,
CA)) to provide cells (eg, EBNA-based systems)
.

It is within the scope of the present invention to use a binding sequence-repressor protein pair in the design of the helper virus and cell lines of the invention, said pair preventing the use of packaging signals in the helper virus genome, Alternatively, it facilitates signal excision, thereby preventing helper virus packaging. Helper adenovirus and producer cell lines are useful in the production of high levels of PAV, allowing preferential packaging of the PAV genome into gene transfer vectors as compared to helper virus, thereby contaminating the helper virus. A means is provided for obtaining a helper-dependent PAV in which is minimized.

The present invention also relates to a method for producing PAV in high yield using the helper virus and the producer cells of the present invention. In such a manner, PAVs occur preferentially, resulting in an enriched formulation. To obtain PAV stocks, use standard methods of molecular biology (eg, issued US patent application Ser. No. 08/8951).
No. 94 and US Pat. No. 5,670,488, incorporated herein by reference), adenovirus 5′ITR and packaging signals and 3′IT.
A PAV genome containing one or more transgenes up to 36 kb in size containing R and operably linked to expression control sequences can be created and placed in a plasmid. The DNA fragment containing the PAV genome is then enzymatically excised from the plasmid and co-transfected with the helper virus of the invention into the producer cells of the invention. According to the invention, PAVs are preferentially packaged. This is because the PAV genome contains a wild-type packaging signal, in contrast to a helper virus in which the packaging signal has been disabled or deleted. When the PAV genome is carried on a plasmid and delivered to a producer cell, such a plasmid can be introduced into the cell lines of the invention by nucleic acid transfer methods including, but not limited to, transfection, lipofection, and electroporation. . Cell lines can be infected with adenovirus helpers that can be used to provide the adenovirus proteins necessary to generate and package the PAV genome. In a preferred embodiment of the present invention, Profec
2-20 μg of DNA encoding the PAV genome is delivered to the cells by lipofection using a kit such as tin (Promega, Madison, WI), and the cells are expressed using a multiplicity of infection (MOI) of 0.5 to 10 according to the invention. Infect with helper virus.

In another aspect of the invention, the PAV producer cell line comprises an integrated PAV genome and nucleic acids encoding DNA binding and / or repressor proteins, such that the PAV genome is conditionally excised. And a nucleic acid encoding a binding protein and / or a repressor protein can be conditionally expressed. In a preferred embodiment, the PAV vector genome adjacent to the lox nucleotide sequence is ligated with Cre recombinase (Sternberg et al., LM
ol.Biol. 150: 467-486, 1981; Sauer et al., Meth.Enzymol. 225: 890-910,
1993) and stably incorporate into a PAV producer cell line that has been treated to express a nucleic acid encoding a repressor protein. Both Cre recombinase and repressor protein are produced inductively using one or more inducible promoters that are susceptible to induction by drugs, such as, inter alia, tetracycline or ecdysone. Infection by the helper virus of the invention, and induction of recombinases and repressors, excise the PAV genome and make it available for replication and packaging by the helper virus, while helper viruses are recruited by the repressor protein for packaging. Disabled. This method of producing PAV only requires infection of the producer cells with a helper virus and induction of recombinase and repressor proteins to obtain a preferentially packaged PAV stock. The use of other site-specific recombinases is also within the scope of this embodiment of the invention, including, for example, FLP recombinase and its target sequence FRT. PA from the cell lines of the present invention by standard methods of virus purification known to those skilled in the art.
V can be purified. For example, the virus in a cell lysate of a producer cell can be purified by a standard CsCl gradient. PAV particles are less dense than the helper virus, form bands at higher gradients, and will be isolated and recovered directly. Alternatively, chromatographic methods,
For example, PAV purification can be performed as indicated in published PCT application WO 97/08298, which is incorporated herein by reference. The PAV yield is calculated by measuring the DNA and protein composition of the purified preparation. Maizel et al. (Virology 36: 115-125, 1968) reported that adenovirus had 13% D
It was determined that it contained NA and the rest was protein. Infecting 293 cells with a PAV helper and then monitoring the transgene activity of the PAV preparation by determining infectious particles using antibodies to the transgene expression product (protein) encoded by PAV. it can. Alternatively, the enzyme activity encoded by the transgene can be measured, for example, by an ELISA assay (eg, β-galactosidase, α-galactosidase, α-antitrypsin). The ability of PAV to enter cells is determined by measuring the amount of viral capsid that binds to cells using an anti-adenovirus antibody. Invasion of the PAV genome into cells can be shown by fluorescence in situ hybridization (FISH). With the novel helper virus of the present invention, minimal contamination of the PAV stock is expected. If there has been helper virus production, it can be marked, for example, by a standard plaque assay on 293 cells. Preferably, the ratio of PAV to helper virus will be greater than 10,000 to 1. The practice of the present invention, unless otherwise specified, includes protein chemistry, molecular virology, microbiology,
Using recombinant DNA techniques, and conventional methods of pharmacy, they are within the skill of the art. Such methods are explained fully in the literature. For example, Current Prot
ocols in Molecular Biology, Ausbel et al., eds., John Wiley & Sons, Inc.
, New York, 1995 and Remington's Pharmaceutical Sciences, 17th ed., Mac
See k Publishing Co., Easton, PA, 1985. The present invention is further described by the following specific examples, which do not limit the scope of the invention in any way.

Example 1 Construction of FRT-Containing Helper AdenoviruspAD / FRT (AV-AVI-AVII) ₂ FRT and pAD / FRT (
AV-AVI-AVI) ₃ Construction of FRT. Plasmid pAd / ITR (1-1
94) Mlu_TwoWas digested with SpeI and MluI. Two copies of AV-AVI
An oligonucleotide containing a 5 'to 3' FRT site upstream of the AVII packaging sequence
Otide is annealed and pAD / ITR (1-194) Mlu_TwoSpeI / Ml
Ligation into the uI site. The obtained vector was used for pAD / FRT (AV-AVI-A
VII)_TwoIt was named. Includes a second FRT site in the same direction as the first FRT site
Oligonucleotide 4758 (5'-CGC GTG AAG TTC CTA TTC CGA AGT TCC TAT
 TCT CTA GAA AGT ATA GGA ACT TCA) (SEQ ID NO: 12) and oligonucleotides
Pide 4759 (5 '-CGC GTG AAG TTC CTA TAC TTT CTA GAG AAT AGG AAC TTC G
GA ATA GGA ACT TCA) (SEQ ID NO: 13) was annealed to obtain pAD / FRT (AV
-AVI-AVII)_TwoLigated into the MluI site of pAD / FRT (AV-A
VI-AVII)_TwoIt was named FRT (FIG. 7). Alternatively, a second FRT site
47 containing a third copy of AV-AVI-AVII upstream of
60 (5 '-CGC GTC GCG TAA TAT TTG TCT AGG GCC GCG GGG ACT TTG ACC GTT T
AG AAG TTC CTA TTC CGA AGT TCC TAT TCT CTA GAA AGT ATA GGA ACT TCA)
Column number: 14) and oligonucleotide 4761 (5'-CGC GTG AAG TTC CTA
 TAC TTT CTA GAG AAT AGG AAC TTC GGA ATA GGA ACT TCT AAA CGG TCA AAG TCC
 CCG CGG CCC TAG ACA AAT ATT ACG CGA) (SEQ ID NO: 15) was annealed and p
AD / FRT (AV-AVI-AVI)_TwoLigated into the MluI site of pAD
/ FRT (AV-AVI-AVI)_ThreeIt was named FRT (FIG. 7).PAD / FRT using oligonucleotides containing 12 copies of AVI ( AVI) Construction of ₁₂ / luc / FRT . AVI package with head and tail joined together
Oligonucleotide containing a 5 'to 3' FRT site upstream of 12 copies of the
Otide is annealed and pAD / ITR (1-194) Mlu_TwoIn the MluI site of
To pAD / FRT (AVI)₁₂I got pGL3 control vector (Promeg
a, Madison, WI) was digested with MluI and BamHI and the SV40 promoter
2427b containing luciferase cDNA under control of enhancer element
The fragment of p was isolated. An oligonucleotide containing a second FRT site is
Neil and pAD / FRT with 2427 bp luciferase fragment
(AVI)₁₂At the MluI site. The resulting vector is (AVI) ₁₂ / Luc cassette containing the FRT adjacent to the pAD / FRT (AVI) ₁₂ / Luc / FRT (FIG. 9).PAD / FRT using an oligonucleotide containing one copy of AVI (AVI ₁₂ ) Construction of FRT . Oligonucleotide 4755 (5'-TCG ACC GCG GGG ACT T
TG ACC) (SEQ ID NO: 16) and 4754 (5 '-TCG AGG TCA AAG TCC CCG CGG)
SEQ ID NO: 16) and oligonucleotide 4754 (5′-TCG AGG TCA AAG TCC CCG CGG) (SEQ ID NO:
: 17) in the presence of T4 DNA ligase
Construct a copy of the AVI packaging sequence with the head and tail joined together
Was. After ligation, the reaction was digested with XhoI to link the heads and tails together.
The product was removed and pSL1180 (Amersham Pharmacia Biotech, Piscataway) was removed.
, NJ) into the XhoI site of pSL / (AVI)₁₂I got AVI anti
The FRT site adjacent to the repeat was inserted in two consecutive cloning steps.
Was. pSL / (AVI)₁₂Was digested with SpeI and partially digested with XhoI. FR
Oligonucleotide HR100 containing T site (5'-CTA GTG AAG TTC CTA TTC C
GA AGT TCC TAT TCT CTA GAA AGT ATA GGA ACT TCC) (SEQ ID NO: 18) and
Oligonucleotide HR101 (5'-TCG AGG AAG TTC CTA TAC TTT CTA GAG A
AT AGG AAC TTC GGA ATA GGA ACT TCA) (SEQ ID NO: 19) (AVI)₁₂upon
It was ligated into the SpeI / XhoI site of the stream. The resulting vector pSL / FRT (
AVI)₁₂Was digested with MluI and partially digested with XhoI. The second FRT site
Oligonucleotide containing HR102 (5'-TCG AGG AAG TTC CTA TTC CGA AGT T
CC TAT TCT CTA GAA AGT ATA GGA ACT TCA) (SEQ ID NO: 20) and oligonucleotide HR103 (5′-CGC GTG AAG TTC CTA TAC TTT CTA GAG A
AT AGG AAC TTC GGA ATA GGA ACT TCC) (SEQ ID NO: 21) (AVI)₁₂Under
It was ligated into the SpeI / XhoI site of the stream. The resulting vector was transformed into pSL / FRT
(AVI)₁₂It was named FRT. Helper containing the above design of 5 'adenovirus packaging signal region
Adenovirus in vitro using standard ligation techniques, or
Constructed by homologous recombination in vivo with the desired adenovirus.

Example 2: Construction of a helper adenovirus containing a lambda operator site Helper vector Ad (AV-AVI-AVII). Adenovirus nucleotides 1 to 194 were isolated by PCR and Ad nucleotides 1 to 357
Was incorporated into pAdvage (Genzyme Gene Therapy, Framingham MA). pAdvantage is the Ad2 genome in pBR322 (ΔE1,
E3Δ2.9). The deleted packaging site (AV-AV
The sequence encoding I-AVII) (nucleotides 334-385) was incorporated into the SpeI / MluI site in the vector, flanking the lambda repressor binding site (FIG. 3a; junction sequence is shown in FIG. 3b). Compared to the wild-type vector, this vector had a 40-fold loss of packaging. Helper vector Ad (AV-AV-AV-AVII)
AVI-AVII) ₂ was constructed (FIG. 4a; junction sequence is shown in FIG. 4b). This helper vector contains two copies of the (AV-AVI-AVII) packaging repeat and has packaging efficiency equivalent to the wild type.

Example 3 Construction of TBTP Helper Adenovirus At the RsrII site engineered into the E3 deleted genome, Ad
2 E3 genes (nucleotides 27970-30937) with 1.8 kb EG
Replaced with FP expression cassette (CMV / EGFP / SvpA). Human genome AA
A 5.0 kb HinpII / AccI fragment from the T gene (ghAAT) was incorporated into the AccI site just upstream of the EGFP expression cassette. The size of the completed vector was 36.9 kb (102% of wild type) (FIG. 10).
After recombination with adenovirus sequences from 293 cells, the vector size was approximately 39
. Will be 6 kb (110%).

Example 4: Construction of producer cells expressing FLP recombinase Plasmid pFLPe6. pOG-Flpe6 was purchased from Francis Stewart (EMBL, Heidelberg, G
ermany) (FIG. 16). This gene contains a mutation of the Flp gene,
The mutation makes the encoded protein more stable at 37 ° C. The gene having upstream and downstream regulatory elements was designated pOG44 (Stratagene, La Jolla, CA).
) Was incorporated after the CMV promoter in FIG. 16 (FIG. 16). pSVK / FLPe6. 2. SalI fragment of pOG-Flpe6
The 9 kb pSVK3 (Pharmacia, Piscataway NJ) was integrated directly into the XbaI / SalI site. Plasmid pSVK / FLPe6 contains a eukaryotic promoter (SV
40e) and has the Flpe6 gene under the control of both the prokaryotic promoter (T7) (Figure 14). pCEP / FLPe6. 2.0 kb Xbal / Sal of pOG-Flpe6
The I fragment was converted to a 10.4 kb pCEP-4 using an adapter linker.
(Invitrogen, San Diego, CA) at the Acc65I / XhoI site. Plasmid pCEP / FLPe6 contains FLpe6 under the control of the CMV promoter.
It has a gene. The plasmid is EB for extrachromosomal maintenance.
It carries the NA-1 gene and the EBV replication origin, and further has the hygromycin gene for plasmid selection (FIG. 15). pTRE / FLPe6. 2.0 kb Xbal / Sal of pOG-Flpe6
The I fragment was integrated into the Acc65I / XhoI site of a 3.1 kb pTRE plasmid (Clontech, Palo Alto, Calif.) Using an adapter linker. The Flpe6 gene in pTRE / FLPe6 contains a minimal CMV promoter (h
CMV ^* ) and operator site (tetO, 1 to 7). Producer cells pCEP / Flpe6 cells. 293 cells and PER. C6 cells, 10-2
Transfected with 0 μg of pCEP / Flpe6 DNA. Following hygromassin selection, cells expressing high levels of Flpe6 are selected based on a functional assay for FLP activity, such as FRT-mediated excision of the target sequence.
Cells with stable extrachromosomal plasmid copy number are selected for PAV amplification. pSVK / Flpe6 cells. Cells are transfected as above with the addition of a plasmid carrying the neo resistance gene marker. Following neo selection, cells expressing high levels of Flpe6 are selected based on a functional assay for FLP activity, such as FRT-mediated excision of the target sequence. PS stably integrated
Cells with VK / Flpe6 DNA are selected for PAV amplification. pTRE / Flpe6 cells. PTK / hygr for transfection
o Add the plasmid (Clontech, Palo Alto, CA) and transfect the cells as described above. Following hygromycin selection, cells expressing low levels of background and high levels of Flpe6 induced by tetracycline are selected based on functional assays for FLP activity, such as FRT-mediated excision of target sequences. Cells with stably integrated pTRE / Flpe6 DNA are selected for PAV amplification.

Example 5: Propagation of PAV with helper adenovirus Semi-confluent P with PAV DNA excised from skeletal DNA (4-20 μg)
ER. Transfect C6 or 293 cells. To select for PAV packaging using a helper vector, the cell expresses a repressor protein that, when bound to a cognate sequence in the helper vector, prevents its packaging. After overnight incubation, cells are infected at an MOI of 1-10 with an E1-deleted helper adenovirus having the appropriate packaging impairment sequence. After observing the complete cytopathic effect, the cells and lysate are collected and the lysate is used for a series of PAV expansions. After several rounds of amplification, the cell lysate is converted to the CsCl band to isolate PAV.

[0034]

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic of a method for producing a packaging defective helper adenovirus.

FIG. 2 is a schematic of the FLP recombinase / FRT Hell-system for excision of packaging sequences from helper adenovirus.

FIG. 3a is a schematic of a helper adenovirus containing a packaging signal adjacent to a lambda operator binding sequence, and FIG. 3b shows the junction structure of the packaging signal sequence and the operator sequence.

FIG. 4a is a schematic of a helper adenovirus containing a packaging signal adjacent to a lambda operator binding sequence, and FIG. 4b shows the junction structure of the packaging signal sequence and the operator sequence.

FIG. 5 is a schematic of a helper adenovirus containing a packaging signal adjacent to a lambda operator binding sequence.

FIG. 6 is a schematic of a helper adenovirus containing a packaging signal adjacent to a lambda operator binding sequence.

FIG. 7 is a schematic of a series of helper adenoviruses containing a packaging signal flanked by FRT binding sequences.

FIG. 8 is a diagram of Ad2HelpFRT containing a packaging signal adjacent to the FRT binding sequence.

FIG. 9 is a diagram of Ad2HelpFRTluc containing a packaging signal and a luciferase marker gene flanked by FRT binding sequences.

FIG. 10 is a schematic of the helper adenovirus TBTP.

FIG. 11 is a schematic of a helper adenovirus with internal ITR sequences that facilitate viral replication.

FIG. 12 is a diagram of the plasmid pTRE / FLPe6.

FIG. 13a is a schematic diagram of a tetracycline induction system for FLPe6 expression, and FIG. 13b shows a tetracycline induction system for FLPe6 expression in 293-tet-off cells.

FIG. 14 is a diagram of the plasmid pTRE / FLPe6.

FIG. 15 is a diagram of the plasmid pCEP / FLPe6.

FIG. 16 is a diagram of the plasmid pOG / FLPe6.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), AU, CA, JP, US (72) Inventor Helen Romantzack 3101 Windsor Ridge Drive, Westborough, Mass. 01581, United States of America (72) Richard Jay Gregory, United States 01866 Westford, Mass., USA Winter Green Rain No. 2 (72) Inventor Donna Amentano 352 Brighton Street Belmont, Mass. 02718 USA F-term (reference) 4B024 AA20 BA80 CA04 DA02 DA20 EA02 FA02 GA11 HA 01 4B065 AA90X AA95X AA95Y AB01 BA02 CA60

Claims (25)

[Claims] 1. An adenovirus genome having a nucleic acid containing a DNA-binding protein and / or a repressor protein binding sequence inserted in or near a packaging signal region, and the binding of the protein to the binding sequence is a helper. A helper adenovirus that facilitates the production of a pseudo-adenovirus vector that interferes with virus packaging. 2. The helper adenovirus of claim 1, wherein the binding sequence comprises a nucleic acid encoding a lambda operator sequence. 3. The helper virus of claim 2, wherein the binding sequence comprises SEQ ID NO: 1. 4. The FLP wherein the binding sequence is recognized by the FLP recombinase
The helper adenovirus according to claim 1, which comprises a recombinant target sequence (FRT). 5. A helper adenovirus which facilitates the production of a pseudo-adenovirus vector comprising an adenovirus genome containing a packaging signal region having one AV-AVI-AVII packaging element repeat. 6. A helper adenovirus which facilitates the production of a pseudo-adenovirus vector comprising an adenovirus genome containing a packaging signal region having two copies of an AV-AVI-AVII packaging element repeat. 7. A helper adenovirus that facilitates the production of a pseudo-adenovirus vector comprising an adenovirus genome containing a packaging signal region having three copies of an AV-AVI-AVII packaging element repeat. 8. A helper adenovirus that facilitates the production of a pseudo-adenovirus vector comprising an adenovirus genome containing a packaging signal region having 12 copies of an AVI packaging element repeat. 9. The helper adenovirus of claim 5, further comprising a nucleic acid containing a lambda operator binding sequence inserted adjacent to a packaging signal in the adenovirus genome. 10. The helper adenovirus of claim 5, further comprising a nucleic acid containing a FLP recombinant target sequence (FRT) inserted at a position adjacent to a packaging signal in the adenovirus genome. 11. The helper adenovirus of claim 6, further comprising a nucleic acid containing a lambda operator binding sequence inserted adjacent to a packaging signal in the adenovirus genome. 12. The helper adenovirus of claim 6, further comprising a nucleic acid containing a FLP recombinant target sequence (FRT) inserted at a position adjacent to a packaging signal in the adenovirus genome. 13. The helper adenovirus of claim 8, further comprising a nucleic acid containing a lambda operator binding sequence inserted at a position adjacent to a packaging signal in the adenovirus genome. 14. The helper adenovirus of claim 8, further comprising a nucleic acid containing a FLP recombinant target sequence (FRT) inserted at a position adjacent to a packaging signal in the adenovirus genome. 15. A producer cell line for producing a pseudo-adenovirus vector comprising and expressing a stably integrated nucleic acid encoding a DNA binding protein and / or a repressor protein. 16. The producer cell line according to claim 15, wherein the DNA binding protein is a lambda repressor. 17. The DNA binding protein is FLP recombinase.
Producer cell line. 18. The producer cell line of claim 15, which is a 293 cell line. 19. PER. 16. The producer cell line of claim 15, which is a C6 cell line. 20. TetR / VP16 operably linked to an expression control sequence.
The producer cell line of claim 18, wherein the nucleic acid encoding is stably integrated into the genome of the cell line. 21. The producer cell line of claim 20, further comprising a stably integrated nucleic acid encoding a FLP recombinase operably linked to an expression control sequence comprising a tetracycline transcriptional regulatory element (TRE). 22. A producer cell line containing and expressing a stably integrated nucleic acid encoding a FLP recombinase operably linked to an expression control sequence comprises a pseudo-adenovirus vector genome (PAV) and an adenovirus genome. A helper adenovirus (in the adenovirus genome, a binding sequence containing a FLP recombination target sequence (FRT) sequence is inserted at a position adjacent to the packaging signal, whereby the helper adenovirus packaging signal is removed from the FLP recombinase; ), And then isolating the PAV vector produced in the cell line. 23. The introduction of one or more of up to 36 kb in size, wherein the PAV genome is operably linked to an adenovirus genome comprising 5 ′ and 3 ′ ITR sequences, adenovirus packaging signals and expression control sequences. 23. The method of claim 22, comprising a gene. 24. TetR / VP16 operably linked to an expression control sequence.
FLP comprising a stably integrated nucleic acid encoding a fusion protein and operably linked to an expression control sequence containing a tetracycline transcriptional regulatory element (TRE)
The producer cell line, which further comprises the stably integrated nucleic acid encoding the recombinase, is ligated to a pseudo-adenovirus vector (PAV) and a helper adenovirus containing the adenovirus genome (in which the FLP recombination target sequence (FRT) sequence Is inserted at a position adjacent to the packaging signal, whereby the helper adenovirus packaging signal is excised by FLP recombinase) and then produced in the cell line. A method for producing a pseudo-adenovirus vector, comprising isolating a PAV vector. 25. The introduction of one or more of up to 36 kb in size, wherein the PAV genome is operably linked to an adenovirus genome comprising 5 ′ and 3 ′ ITR sequences, adenovirus packaging signals and expression control sequences. 25. The method of claim 24, comprising a gene.