JP2004513638A - vector - Google Patents

Abstract

The present invention provides a two component system for in vitro cloning of a heterologous polynucleotide into adenovirus DNA. The first component is an insertion donor that contains a heterologous polynucleotide encoding a heterologous polypeptide. The second component is a vector donor containing the adenovirus genome and expression cassette. Insert and vector donors are phage lambda-derived recombination sites for inserting a heterologous polynucleotide into an expression cassette capable of forming an adenovirus expression clone in vitro in the presence of the appropriate recombinant mediator protein or proteins. For site-specific recombination. The invention also provides a method of making a recombinant adenovirus and the use of the first and second components in the method.

Description

[0001]
Adenoviruses are a group of DNA viruses that can generally cause mild infections of human respiratory disease, conjunctivitis, and gastroenteritis in infants. Adenoviruses have been extensively studied for many years because of their easy growth in cell culture (eg, RNA splicing was first described in adenovirus-infected cells). The ability of adenovirus to infect human cells with up to 100% efficiency has led to its use as a vector for introducing foreign (recombinant) DNA both in cell culture and in humans (gene therapy). Recombinant adenoviruses generally lack specific regions of DNA (eg, the E1 region required for replication, E3 required for host immune evasion). There are two aspects to this purpose. First, the removal of unnecessary DNA regions provides a space for the introduction of foreign DNA that can be packaged as adenovirus DNA and subsequently introduced into human cells (additional DNA is incorporated into the genome). , There is some room for insertion without affecting infectivity). Second, elimination of the E1 region dictates that the recombinant virus can infect human cells but cannot replicate (this is important in terms of safety). To make and replicate the recombinant adenovirus, the DNA is transfected (ie, deleted by the recombinant adenovirus by the cell line) into a cell line that has the designed E1 region in the genome (eg, HEK293). Replication mechanism is provided).
[0002]
Recombinant adenovirus technology is often based around human adenovirus type 5. The genome of Ad5 is 35.935 kb. This entire sequence was cloned into a plasmid vector and E. coli. However, due to the length of the adenovirus sequences, there are very few suitable restriction enzyme sites available for cloning directly into the vector. To avoid this problem, two vector approaches have been adopted. One vector contains the complete adenovirus sequence minus the E1 and E3 sequences. The second vector contains a eukaryotic promoter (eg, CMV) upstream of the cloning site (the site for insertion of foreign DNA), and a polyadenylation sequence required for stabilization of the transcribed RNA. Adjacent to this expression cassette are two regions of adenoviral DNA in common with the adenoviral sequence of the first vector. Therefore, the expression cassette from the second vector can be introduced into the adenovirus sequence in the first vector by the process of homologous recombination. This tedious, accurate and time-consuming method has traditionally been performed on HEK293 cells. The virus produced must be a plaque that has been purified, propagated, and tested to ensure that the desired recombinant DNA has been introduced.
[0003]
Recent developments in recombinant adenovirus production, the "AdEasy (TM)" system (He, TG et al., PNAS, 95, 1998, 2509-2514) allow limited recombination. Performing the plasmid recombination step in E. coli strain BJ5183 simplified the procedure.
[0004]
Recombinant adenovirus DNA was digested with restriction enzymes and E. coli can be identified by clonal adenovirus DNA prepared in E. coli. The cloned adenovirus DNA is transfected into HEK293 cells to create adenovirus, thereby effectively eliminating the need for plaque purification. This method is currently being commercialized (Q-biogene: Adeasy ™). A further improvement of this system is that the recombinant adenovirus co-expresses the green fluorescent protein from the second CMV promoter. This makes the infection efficient (and thus the gene delivery efficient) and simplifies the assessment. Our experience with the Adeasy ™ system has shown that recombination procedures are difficult. This can include high background from the expression vector, unpredictable recombination events, and low recombination efficiency. Therefore, there is a need for further improvement in recombinant adenovirus production.
[0005]
According to one aspect of the present invention,
into an adenovirus DNA containing i) a first component that is an insertion donor containing a heterologous polynucleotide encoding a heterologous polypeptide, and ii) a second component that is a vector donor containing an adenovirus genome and an expression cassette. A two-component system for in vitro cloning of heterologous polynucleotides of
In this system, the insertion and vector donors are site-specific for inserting a heterologous polynucleotide into an expression cassette capable of forming an adenovirus expression clone in vitro in the presence of a suitable recombinant mediator protein or proteins. Applicable for recombination.
[0006]
Preferably, site-specific recombination uses a recombination site from phage lambda. For a review of the recombination of λ, the reader is referred to the following: Landy (1989) Ann Rev Biochem 58, 913 and Ptashne (1992) A Genetic Switch, Cell Press, Cambridge. The reader is referred to US Pat. No. 5,888,732 (Life Technologies) for further technical details of site-specific recombination using insert and vector donor moieties. In use, the recombination reaction is elevated such that the recombinant mediator protein cleaves left and right of the heterologous polynucleotide in the insert donor and is ligated into the vector donor, thereby forming an adenovirus expression clone. Specific cleavage and ligation reactions occur.
[0007]
Preferably, the expression cassette comprises a polynucleotide encoding a fluorescent protein downstream of an internal ribosome entry site for expression from the same mRNA as the heterologous polypeptide.
[0008]
Preferably, the systems described herein include at least one of the following:
i) a CMV promoter in the expression cassette;
ii) replication defective adenovirus type 5 genome;
iii) a vector donor containing the ccdB gene;
iv) an inserted donor containing a selectable marker;
v) a vector donor containing a selectable marker;
vi) Site-specific recombination sites based on either phage λattB with attP or phage λattL with attR.
[0009]
More preferably, the system comprises all of the elements i) to vi). The attBxattP reaction is mediated by the proteins Int and IHF (Clonase BP ™, Life Technologies). The attL × attR reaction is mediated by the proteins Int, IHF and Xis (Clonase LR ™, Life Technologies). Int and Xis are from λ; IHF is from E. coli. E. coli. Here, “x” indicates recombination. Engineered recombination sites that confer efficient or specific advantages over the wild-type sequence as described in US Pat. No. 5,888,732 are also contemplated.
[0010]
According to another aspect of the present invention,
i) In vitro, a first component as defined herein and a second component as defined herein, in the presence of a suitable recombinant mediator protein or proteins, to form an expression clone. Mixing with
ii) transformation of the product from i) into a host organism suitable for expression clone replication,
iii) transfection of the product from ii) into a suitable host for adenovirus replication,
A method for producing a recombinant adenovirus is provided.
[0011]
Preferably, the method uses the first and second components as defined in elements i) to vi) above, and the host organism for replication of the expressed clone is E. coli. coli, and the host organism for adenovirus replication is HEK293 cells.
[0012]
According to another aspect of the present invention, there is provided a second component as defined herein.
[0013]
According to another aspect of the present invention, there is provided the use of a first component as defined herein in a method as defined herein.
[0014]
According to another aspect of the present invention, there is provided use of a second component as defined herein in a method as defined herein.
[0015]
The present invention is illustrated by the following non-limiting examples:
FIG. 1 shows a vector donor, where 1 = fluorescent protein, 2 = IRES element, 3 = attR2, 4 = ccdB + cm ^R , 5 = attR1, 6 = adenovirus sequence (ΔE1 / ΔE3), 7 = Origin of replication, 8 = ampicillin resistance, 9 = restriction enzyme site for excision of plasmid backbone, 10 = adenovirus LITR sequence, and 11 = SV40PA;
[0016]
FIG. 2 shows the inserted donor, where 1 = gene of interest, 2 = attL2, 3 = kanamycin resistance, 4 = origin of replication, 5 = attL1;
[0017]
FIG. 3 shows the in vitro cloning of lacZ into adenovirus expression clones, wherein:
A = insert donor (where 1 = gene of interest (lacZ), 2 = attL2, 3 = Kn ^r , 4 = ori, 5 = attL1),
B = vector donor (where 1 = fluorescent protein, 2 = IRES, 3 = attR2, 4 = ccdB, 5 = attR1, 6 = CMV promoter, 7 = adenovirus sequence, 8 = ori, 9 = Amp ^r , 10 = SV40PA),
C = expression clone (Amp ^r ) (where 1 = fluorescent protein, 2 = IRES, 3 = AttB2, 4 = lacZ, 5 = AttB1, 6 = CMV promoter, 7 = adenovirus sequence, 8 = ori, 9 = Amp ² , 10 = SV40PA),
D = Bi- product (kana ^{r) (where, 1 = ccdB, 2 = AttP2,3} = Kn r, 4 = ori, 5 = AttP1),
E = Int, IHF, Xis protein,
F = on ampicillin or E.C. G.E. does not grow in E. coli DH5α (ccdB lethal). E. coli (eg, DH5α), clones are identi? ed by selection on ampicillin plates, and transfected into HEK293 cells to generate the virus.
General, molecular biology techniques are described in "Current Protocols in Molecular Biology" Volumes 1-3, edited by FM Asubel, R Brent and RE Kingston, published by John Wiley, 1998.
[0019]
Example 1
We used an in vitro cloning system to make recombinant adenovirus production easier and more efficient. In addition, we incorporated a fluorescent protein (Matz et al (1999) Nat. Biotechnol. 17, 969-973; Lukyanov et al (2000) J. Biol. Chem. 275, 2587-25882) as an infection marker.
[0020]
Briefly, an expression cassette containing the complete adenovirus genome (excluding E1 and E3); CMV promoter attR1-chloramphenicol resistance-ccdB-attR2, internal ribosome binding site (IRES), fluorescent protein sequence and SV40 poniadenylated sequence. , And a vector was created.
[0021]
This vector is a vector donor. Any sequence in the insert donor can be effectively cloned directly into this vector donor via in vitro recombination. E. FIG. Background is reduced to zero due to ccdB genotoxicity in E. coli (the vector donor DNA is increased in E. coli strain DB3.1, which has a gyrA mutation and is resistant to ccdB). Therefore, in vitro integration into the vector donor by the gene of interest in the insert donor, followed by E. coli used for vector donor selection. Transformation of E. coli DH5a (vector donor ampicillin resistance, insertion donor kanamycin resistance) produces only recombinant adenovirus DNA due to the toxicity of the ccdB gene. The DNA is then digested, the plasmid backbone is removed, and transfected directly into HEK293 cells to create a recombinant virus.
[0022]
We have used this system to create recombinants expressing lacZ from an inserted donor containing lacZ. It is quick and efficient, and we believe it has significant advantages over current AdEasy systems. These constructs also express fluorescent proteins from IRES elements. This means that they are expressed from the same transcribed messenger RNA as the recombinant gene. Using "AdEasy ™", green fluorescent protein is expressed from a second, separate CMV promoter. In this case, expression of the fluorescent protein does not guarantee recombinant gene transcription.
[0023]
After the sequence encoding six histidine residues, E. coli A 3168 base pair fragment containing the entire coding region of the E. coli lacZ gene was isolated from pZeoSV2 / lacZ (Invitrogen) by restriction enzyme digestion (NcoI and EcoRI). The lacZ DNA fragment was separated from the plasmid backbone by gel electrophoresis, excision from the gel, and purification using a Geneclean ™ Spin Kit. The isolated fragment was then cloned into NcoI and EcoRI of the insert donor pENTR11 (Life Technologies). Briefly, pENTR11 was digested with NcoI and EcoRI, purified by gel excision, and dephosphorylated with shrimp alkaline phosphatase (Roche). The lacZ fragment was then ligated into pENTR11 in vitro using T4 DNA ligase (Roche) and E. coli DH10B electrocompetent cells (Life Technologies). After overnight growth on kanamycin plates, the pENTR11 clone containing lacZ was identified by restriction digestion of plasmid DNA. Therefore, this clone contained the lacZ coding sequence flanked by one bacteriophage attL1 and attL2 sites. For the generation of adenovirus DNA containing the lacZ gene, a vector donor containing the following was prepared:
(I) Adenovirus DNA sequence (excluding E1 and E3 regions)
(Ii) Excisable plasmid backbone (ampicillin resistance)
(Iii) CMV promoter for the expression of heterologous genes in mammalian cells. (Iv) The chloramphenicol resistance gene flanked by 1 bacteriophage attR1 and attR2 sites and E. coli. coliccdB gene (v) internal ribosome binding site (IRES) element (vi) fluorescent protein coding sequence (vii) SV40 polyadenylation sequence for mRNA stability.
[0024]
This vector donor was incubated with the insert donor (pENTR11 / lacZ) in a buffer containing the recombinant mediator protein, Int, IHF, and Xis (LR clones ™, Life Technologies). The obtained DNA was used as E. coli. coli DH10B electrocompetent cells and plated on ampicillin plates. Fifteen clones were selected among the thousands present and analyzed by restriction digest of the prepared plasmid DNA. Of these, 14 (93%) contained the desired adenovirus lacZ construct and only 1 did not.
[0025]
For production of adenovirus expressed lacZ and fluorescent proteins, adenovirus lacZ plasmid DNA was digested with PacI, the plasmid backbone was removed, and transfected into HEK293 using Lipofectamine ™ (Life Technologies). After 12 days of incubation, virus growth was evident and virus was recovered from the cell lysate.
[0026]
Comparative Example 1
E. FIG. Recombinant adenovirus DNA production using "AdEasy ™" homologous recombination in E. coli BJ5183 was less production and efficient than the in vitro method of Example 1. In general, significantly fewer colonies were obtained (less than 20 per transfection compared to thousands) and these few colonies were the desired recombinants (typically 10-30%).
[Brief description of the drawings]
FIG. 1 shows a vector donor.
FIG. 2 shows an inserted donor.
FIG. 3 shows in vitro cloning of lacZ into adenovirus expression clones.

Claims (10)

A two component system for in vitro cloning of a heterologous polynucleotide into adenovirus DNA, comprising:
i) a first component that is an insertion donor that includes a heterologous polynucleotide encoding a heterologous polypeptide, and ii) a second component that is a vector donor that includes an adenovirus genome and an expression cassette, wherein:
Insert and vector donors are adapted for site-specific recombination to insert a heterologous polynucleotide into an expression cassette capable of forming an adenovirus expression clone in vitro in the presence of the appropriate recombinant mediator protein or proteins. Be done
The system. 2. The system of claim 1, wherein the site-specific recombination uses a recombination site from phage lambda. The system of claim 1 or 2, wherein the expression cassette comprises a polynucleotide encoding a fluorescent protein downstream of the internal ribosome binding site for expression from the same mRNA as the heterologous polypeptide. 4. The system of any of claims 1 to 3, comprising at least one of the following:
i) a CMV promoter in the expression cassette;
ii) replication defective adenovirus type 5 genome;
iii) a vector donor containing the ccdB gene;
iv) an inserted donor containing a selectable marker;
v) a vector donor containing a selectable marker;
vi) Site-specific recombination sites based on either phage λattB with attP or phage λattL with attR. 5. The system of claim 4, comprising all of said elements i) to vi). A method for producing a recombinant adenovirus, comprising:
i) the first component of any of claims 1-5 in the presence of a suitable recombinant mediator protein or proteins so as to form an expression clone; the second component of any of claims 1-5; Mixing in vitro with
ii) transformation of the product from i) into a host organism suitable for expression clone replication,
iii) transfection of the product from ii) into a suitable host for adenovirus replication,
The method comprising: The first and second components are as described in claim 5, wherein the host organism for expression clone replication is E. coli. 7. The method of claim 6, wherein the host organism is E. coli and the host organism for adenovirus replication is HEK293 cells. A second component according to claim 1. Use of the first component according to any of claims 1 to 5 in a method according to claim 6 or 7. Use of the second component according to any one of claims 1 to 5 in a method according to claim 6 or 7.