阅读说明：本技术 用于逆转录病毒产生的稳定细胞系 (The stable cell lines generated for retrovirus ) 是由 C.文克 于 2018-04-18 设计创作，主要内容包括：本发明涉及逆转录病毒生产细胞,其包含编码以下的核酸序列：gag和pol蛋白；包膜蛋白或其功能性替代物；可扩增选择标记；和逆转录病毒载体颗粒的RNA基因组；其中所述核酸序列全部整合于逆转录病毒生产细胞基因组内的单一基因座处。本发明还涉及包含非哺乳动物复制起点且能够容纳至少25千碱基(kb)的DNA的核酸载体,其特征在于所述核酸载体包含编码以下的逆转录病毒核酸序列：gag和pol蛋白,和env蛋白或其功能性替代物。所述核酸载体另外包含编码可扩增选择标记的核酸序列。本发明还涉及使用所述核酸载体以产生稳定的逆转录病毒包装和生产细胞系的用途和方法。(The present invention relates to retroviral producer cells, and it includes encode nucleic acid sequence below: gag and pol albumen；Envelope protein or its functional replacement；Amplifiable selected marker；With the rna gene group of retroviral vector particle；Wherein the nucleic acid sequence is all integrated at the term single gene seat in retroviral producer cells genome.The invention further relates to comprising nonmammalian replication orgin and can accommodate at least 25 kilobase (kb) DNA nucleic acid carrier, it is characterized in that the nucleic acid carrier includes to encode retroviral nucleic acid sequence below: gag and pol albumen and env albumen or its functional replacement.The nucleic acid carrier additionally comprises the nucleic acid sequence for encoding amplifiable selected marker.The invention further relates to use the nucleic acid carrier to generate the purposes and method of stable retrovirus packaging and production cell line.)

1. retrovirus incasing cell, it includes the coding nucleic acid sequences below as single expression construct:

Gag and pol albumen；

Env albumen or its functional replacement；With

Amplifiable selected marker；

Wherein the expression construct is all incorporated into one at the individual gene seat in retrovirus incasing cell genome It rises and is used as unit；And

Wherein the copy number of the unit is 2 or more.

2. the retrovirus incasing cell of claim 1, wherein the amplifiable selected marker is dihyrofolate reductase (DHFR) or glutamine synthelase (GS).

3. the retrovirus incasing cell of claim 2, the amplifiable selected marker described at this time is DHFR, wherein including coding The expression construct of the nucleic acid sequence of DHFR includes the nucleic acid sequence of SEQ ID NO:1.

4. the retrovirus incasing cell of any one of claims 1 to 3, wherein the unit further includes expression building Body, the expression construct include the nucleic acid sequence of encoding selectable markers.

5. the retrovirus incasing cell of any one of claims 1 to 4, wherein the unit further includes expression building Body, the expression construct include coding auxiliary generevOr function is similar to the nucleic acid sequence of gene or function similar system.

6. the retrovirus incasing cell of any one of claims 1 to 5, wherein encoding gag and pol albumen, env albumen or The nucleic acid sequence or auxiliary gene of its functional replacementrevOr function is similar to the nucleic acid sequence source of gene or function similar system From the retrovirus selected from slow virus, α-retrovirus, γ-retrovirus or foamy retrovirus.

7. the retrovirus incasing cell of any one of claims 1 to 6, wherein one of described expression construct or more Kind includes CMV promoter, and preferably wherein the CMV promoter additionally comprises at least one Tet operon.

8. the retrovirus incasing cell of any one of claims 1 to 7 additionally comprises insulator, preferably wherein every There are insulators between a expression construct.

9. the retrovirus incasing cell of any one of claims 1 to 8, wherein the cell is mammalian cell, it is excellent Wherein the mammalian cell is HEK 293T to selection of land.

10. the retrovirus incasing cell of any one of claims 1 to 9, wherein the unit further includes expression structure Body is built, the expression construct includes the nucleic acid sequence of the rna gene group of encoding hiv reverse transcriptase carrier granular.

11. retroviral producer cells, it includes the coding nucleic acid sequences below as single expression construct:

Gag and pol albumen；

Env albumen or its functional replacement；

Amplifiable selected marker；With

The rna gene group of retroviral vector particle；

Wherein the expression construct is all incorporated into one at the individual gene seat in retroviral producer cells genome It rises and is used as unit；And

Wherein the copy number of the unit is 2 or more.

12. comprising nonmammalian replication orgin and can accommodate at least 25 kilobase (kb) DNA nucleic acid carrier, feature It is that the nucleic acid carrier includes to encode nucleic acid sequence below:

Gag and pol albumen,

Env albumen or its functional replacement, and

Amplifiable selected marker,

Wherein every kind of nucleic acid sequence is arranged as the individual expression construct in nucleic acid carrier.

13. the nucleic acid carrier of claim 12, wherein the amplifiable selected marker is DHFR or GS.

14. the nucleic acid carrier of claim 13, the amplifiable selected marker described at this time is DHFR, wherein including the core of encoding D HFR The expression construct of acid sequence includes the nucleic acid sequence of SEQ ID NO:1.

15. the nucleic acid carrier of any one of claim 12 to 14 further includes expression construct, the expression construct Nucleic acid sequence comprising encoding selectable markers.

16. the nucleic acid carrier of any one of claim 12 to 15 further includes expression construct, the expression construct Include auxiliary generevOr it is similar to the nucleic acid sequence of gene or function similar system.

17. the nucleic acid carrier of any one of claim 12 to 16, wherein the nucleic acid carrier is selected from: bacterial artificial chromosome, Artificial chromosome derived from yeast artificial chromosome, P1-, F sticking grain or sticking grain, preferably bacterial artificial chromosome.

18. the nucleic acid carrier of any one of claim 12 to 17 additionally comprises the RNA of encoding hiv reverse transcriptase carrier granular The nucleic acid sequence of genome.

19. the method for generating stable Retronituse encapsulated cell line comprising following steps:

(a) nucleic acid carrier of any one of claim 12 to 18 is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian cell growth；With

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number.

20. the method for generating stable retroviral producer cells system comprising following steps:

(a) nucleic acid carrier of claim 18 is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian host cell growth；With

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number.

21. the method for claim 19 or 20, wherein step (b) and (c) are repeated twice or more times.

22. the method for any one of claim 19 to 21 further comprises selecting tool in culture after step a) There is the mammalian host cell of the nucleic acid sequence encoded on the carrier being integrated into mammalian host cell genome Step.

23. the method for any one of claim 19 to 22, wherein the selective agent is DHFR inhibitor, preferably methotrexate (MTX) (MTX) or GS inhibitor, preferably methionine sulfoxide imines (MSX).

24. passing through any one of claim 19 or any one of the claim 21 to 23 when being subordinated to claim 19 The retrovirus incasing cell that method obtains.

25. the method by any one of claim 20 or the claim 21 to 23 when being subordinated to claim 20 obtains Retroviral producer cells.

26. the method for generating Replication defective retroviral vector particle comprising following steps:

(a) nucleic acid carrier of any one of claim 12 to 18 is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian host cell growth；

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number； With

(d) food in one's mouth selected in further incubation step (c) under conditions of generating Replication defective retroviral vector particle Newborn animal host cell.

27. the method for claim 26 also comprises the step of separating Replication defective retroviral vector particle.

28. the Replication defective retroviral vector particle obtained by the method for claim 26 or claim 27.

Invention field

The present invention relates to the nucleic acid carriers of gene and application thereof needed for generating comprising retroviral vector.It additionally provides The method that cell line was packed/produced to preparation comprising the retrovirus of nucleic acid carrier as described herein.

Background of invention

In gene therapy, by endogenous cell of the genetic material delivery into main body in need for the treatment of.Inhereditary material can will be new Gene introduces main body, introduces the additional copy of pre-existing gene, or be introduced into the different equipotentials for the gene being present in main body Gene or variant.Itd is proposed virus carrier system as gene therapy effective gene delivering method (Verma and Somia (1997) Nature 389: 239-242)。

Specifically, these viral vectors are based on retrovirus family member, because they can be effective by its gene Load is integrated into the genome of host.Retroviral vector is designed to retain packaging and delivering reverse transcription virus gene Those of indispensable protein needed for group, but remove any nonessential auxilin, including cause its disease profile.Retrovirus The example of carrier includes slow virus carrier, such as based on those of human immunodeficiency virus type 1 (HIV-1), since they can It is integrated into non-proliferative cell and is widely used.

Currently, most of viral vectors generate and by viral gene transient cotransfection into host cell line.It uses It is present in the bacterial plasmid in host cell and only introduces viral gene within the limited period, because viral gene is retained in matter On grain and unconformity is into genome.Therefore, the inhereditary material of transient transfection is not transferred to offspring during cell division.

In the presence of several disadvantages relevant to transient transfection method, otherness between such as batch, the high cost of transfection reagent and It is difficult to maintain quality control (referring to Segura et al. (2013)Expert Opin. Biol. Ther. 13(7): 987- 1011).Transfection process itself is also laborious and is difficult to scale up scale.Remove the plasmid impurity left during carrier preparation Task is difficult (referring to Pichlmair et al. (2007)J. Virol. 81(2): 539-47)。

It is related to transiently transfecting in order to solve the problems, such as, it is expected that developing retrovirus packaging and production cell line with simplification Retroviral vector production.

Package cell line is generated by the way that the cell line of retroviral vector can be packed with plasmid transfection, wherein a Other plasmid carries retrovirus packaging gene and unique eukaryon selected marker.Retrovirus packaging gene is integrated into packet In the genome for filling cell line, and it is described as being stably transfected.In past 20 years, various trials have been made to generate Stabilising packaging and production cell line for retroviral vector.

The packaging generated in host cell gene group and production cell line are integrated into via by retroviral vector component In there are many reports.Firstly, it may be laborious and inflexible for successively introducing retroviral vector component.When inverse When transcription vector component is integrated into host cell gene group, there is also its heredity and/or instable ask is transcribed Topic, because integration site is uncertain (Ni etc., (2005)J. Gene Med. 7: 818–834.)。

Being remarkably decreased for viral vectors productivity is also reported during the suspension adaptation of production cell line and increase scale (Farson et al. (2001)Hum. Gene Ther. 12: 981–997;Guy et al. (2013)Hum. Gene Ther. Methods. 24(2): 125-39)。

It also has been made attempting to produce the transfection cell for the recombinant protein for providing reliable high titre in biological-pharmacy System.This class process is usually directed to the cell of screening transfection to identify the optimum growh with recombinant protein and the cell line of generation (for example, with reference to Wurm, 2004,Nature Biotechnology 22, 1393-1398)。

Known method is related to selecting the cell line of stable transfection, and wherein desired recombinant gene has been integrated into host cell Genome in and expand so that the cell line include increase copy number desired recombinant gene.This is by recombinating base with target Because together with being carried out by the amplifiable selectable marker gene cotransfection host cell of known toxicity Drug inhibition.Then, such as it is logical The cell for cultivating transfection in the culture medium containing drug toxicity is crossed, the cell of stable transfection is made to be subjected to the known poison of increasing concentration Property drug.In this manner, can choose population of transfected cells, there is the amplifiable selection for increasing copy number by gene magnification Marker gene causes the expression of amplifiable selected marker to increase, and thus assigns the resistance to drug toxicity.Due to gene Amplification procedure leads to the amplification of amplifiable selected marker and surrounding DNA sequences, in desired recombinant gene in host cell gene group In the case where interior integration adjacent with gene that is encoding amplifiable selected marker, the core of possible coamplification encoding target recombination Acid sequence is together with those of amplifiable selected marker of coding.The process recombinates base dependent on amplifiable selectable marker gene and target Because internal interconnection and integration into host cell gene group (Kaufman, et al. 1985, Mol. Cell. Biol. 5: 1750-1759)。

In view of the packaging for obtaining stable transfection available at present and production cell line for generating retrovirus vector The method of body requires reverse transcription virus gene to be successively integrated into host cell gene group, and the site of each integration event All be it is random and uncertain, so cell line as extremely difficult acquisition, wherein all reverse transcription virus genes have been integrated Near to amplifiable selectable marker gene, to obtain the cell line that wherein reverse transcription virus gene has expanded.It is therefore contemplated that adopting It is selected to generate the cell line of the viral vectors of high titre using amplifiable selected marker with the option program of foundation to be not sound feasible Border.

The method stablized retrovirus packaging therefore, the purpose of the present invention is to provide preparation and produce cell line, gram One or more disadvantages relevant to existing method are taken.

Summary of the invention

The present inventor has developed the new paragon for preparing retrovirus packaging and production cell line, is related to using comprising the non-food in one's mouth Newborn animal coping starting point and the nucleic acid carrier that at least 25 kilobase (kb) DNA can be accommodated, such as comprising retroviral vector The bacterial artificial chromosome of reverse transcription virus gene and amplifiable selectable marker gene necessary to producing.This, which allows to express, generates Reverse transcription virus gene needed for Replication defective retroviral vector particle is to improve ask relevant to transient transfection method Topic.Further, since required reverse transcription virus gene and amplifiable selectable marker gene are all within single nucleic acid carrier transfer Dye is into host cell, so the nucleic acid carrier is integrated into the term single gene seat in host genome.This improves and successively turns It contaminates, cause the problem for integrating reverse transcription virus gene correlation at different locus in host genome, and can with using Selected marker is expanded to expand the problem of the reverse transcription virus gene correlation in the cell.

Comprising nonmammalian replication orgin and the nucleic acid of at least 25kb DNA (that is, large size construct DNA) can be accommodated The use of carrier has the advantages that several.It is possible, firstly, to first in nonmammalian cells (for example, microbial cell, such as bacterium Cell) rather than carrier is operated in mammalian host cell, this is easier to them using (for example, bacterial artificial chromosome Body can operate in Escherichia coli first).Once having prepared nucleic acid carrier, so that it may be introduced into mammalian host cell In, and can choose nucleic acid carrier and be integrated into any mammalian cell in endogenous chromosome to separate stable cell System.

Retroviral nucleic acid introducing mammalian host cell can also be occurred in a single step, help to subtract Pressure and quiet hour range are selected less.This, which allows more rapidly to screen, potentially packs and produces cell and reduce material cost, Because only used single carrier, rather than multiple plasmid vectors are used as prior method.Specifically, by saving matter Grain cost, reduces required Benzonase treating capacity (in slow virus at required transfection reagent (such as polyethyleneimine [PEI]) The amount of DNA is reduced in cutting, it is therefore desirable to which less Benzonase removes the excess in downstream processing) and reduce test Cost (does not need the residual plasmid in test slow virus product), and the use of system reduces production cost at present.

In addition, retrovirus generates necessary reverse transcription virus gene (with or without transfer vector) He Kekuo Increase selectable marker gene to be present in nucleic acid carrier, so that when carrier is introduced into mammalian host cell, retrovirus Gene and amplifiable selectable marker gene all will be at the locus that be integrated into mammalian host cell genome.This It can overcome when such as being integrated at reverse transcription virus gene random integration and the different locus in host cell gene group The problem of gene silencing that may occur.This also offers the packets for generating the retroviral vector particle with high titre Dress or production cell program, wherein the amplification of reverse transcription virus gene independent of reverse transcription virus gene in vivo with can expand Increase the connection of selected marker.

Therefore, according to the first aspect of the invention, retrovirus incasing cell is provided, it includes as independent table The coding of expression constructs nucleic acid sequence below:

Gag and pol albumen；

Env albumen or its functional replacement；With

Amplifiable selected marker；

Wherein the expression construct is all incorporated into one at the individual gene seat in retrovirus incasing cell genome It rises and is used as unit；And

Wherein the copy number of the unit is 2 or more.

In a further aspect of the invention, retroviral producer cells are provided, it includes as single expression The coding of construct nucleic acid sequence below:

Gag and pol albumen；

Env albumen or its functional replacement；

Amplifiable selected marker；With

The rna gene group of retroviral vector particle；

Wherein the expression construct is all incorporated into one at the individual gene seat in retroviral producer cells genome It rises and is used as unit；And

Wherein the copy number of the unit is 2 or more.

In an alternative aspect of the invention, provides comprising nonmammalian replication orgin and at least 25,000 can be accommodated The nucleic acid carrier of the DNA of base (kb), it is characterised in that the nucleic acid carrier includes to encode nucleic acid sequence below:

Gag and pol albumen,

Env albumen or its functional replacement, and

Amplifiable selected marker,

Wherein every kind of nucleic acid sequence is arranged as the individual expression construct in nucleic acid carrier.

In another further aspect of the invention, the side for generating stable Retronituse encapsulated cell line is provided Method comprising following steps:

(a) nucleic acid carrier defined herein is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian cell growth；With

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number.

In an alternative aspect of the invention, it is thin to provide the retrovirus packaging obtained by method described herein Born of the same parents.

In a further aspect of the invention, the method for generating stable retroviral producer cells system is provided, Itself the following steps are included:

(a) nucleic acid carrier defined herein is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian host cell growth；With

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number.

In another further aspect of the invention, it is raw to provide the retrovirus obtained by method described herein Produce cell.

In an alternative aspect of the invention, the method for generating Replication defective retroviral vector particle is provided, Itself the following steps are included:

(a) nucleic acid carrier defined herein is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian host cell growth；

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number； With

(d) food in one's mouth selected in further incubation step (c) under conditions of generating Replication defective retroviral vector particle Newborn animal host cell.

In a further aspect of the invention, provides and reversed by the replication defect type that method described herein obtains Record vector particles.

Attached drawing

Fig. 1: the gradually guide of building BACpack-WTGP-277delU5 and BACpack-SYNGP-277delU5

Fig. 2: stable polyclonal consolidated material is selected.It is transfected using calcium phosphate with BACpackWTGagPol-Transfer HEK293T attached cell.Bleomycin (Zeocin) generates stable consolidated material after selecting 2 weeks.In order to observe stable transfection Whether virus can be generated, with more consolidated materials 48 hours of Doxycycline (Doxycycline) induced stable.After induction 48 hours Harvest vial supernatant, filter by 0.22 μm of filter and pass through transduce HEK293T cell titrated.The transduction of the GFP positive Cell is for calculating transduced unit/ml (TU/mL).

Fig. 3: stable Transfection ofsuspension clone is generated.Use 293fectin reagent BACpackWTGagPol- Transfer transfects HEK293 6E cell.Bleomycin generates stable consolidated material after selecting 2 weeks.It will be steady by limiting dilution Determine consolidated material to be cloned into 96 orifice plates to obtain single cell clone, then be expanded.GFP is detected most by fluorescence microscopy Good clone 1,14,15 and 16, with adhere-wall culture base (DMEM+FBS), then suspension adaptation (FreeStyle culture medium) obtains.

Fig. 4: slow virus is induced in the clone that suspends.In order to observe whether stable HEK6E transfectant can generate virus, It is cloned 48 hours with Doxycycline (2 μ g/ml) induction 20ml stable suspersion.Induction harvested vial supernatant after 48 hours, passed through 0.45 μm of filter filters and passes through transduction HEK293T cell and titrated.It is single that transduction is calculated using GFP positive transduced cells Position/ml (TU/mL).

Fig. 5: according to the vector titer for the clone that embodiment 4 generates.Vector titer as the result is shown from clone 1 and 16 exists 5th generation and the 21st instead of between moderately increase.

Fig. 6: building BACpack-WTGP-DHFR-277delU5 and BACpack-SYNGP-DHFR-277delU5 is gradually Guide.

Fig. 7: includedhfrThe nucleic acid sequence of the expression construct of gene.

Fig. 8: the gradually guide of building BAC2.0 GS.

Detailed description of the invention

Definition

Unless otherwise defined, otherwise all technical and scientific terms used herein has and technology people of the art The normally understood identical meaning of member.The all patents and publications being mentioned above all is passed through reference and is integrally incorporated with it.

Term " include (comprising) " cover " including (including) " or " by ... form (consisting) ", For example, the composition of " including (comprising) " X can only be made of X or may include additional something, such as X+Y.

The scope limitation of feature is specific material or step and will not substantive shadow by term " substantially by ... form " Ring claimed those of the fundamental characteristics of feature.

Term " consist of " excludes the presence of any additional component.

Term " about " about numerical value x refers to such as x ± 10%, 5%, 2% or 1%.

Term " carrier " or " nucleic acid carrier " are to refer to manually carry external (i.e. external source) inhereditary material to another Medium in cell can be replicated and/or be expressed in cell.The example of carrier includes nonmammalian nucleic acid carrier, Such as bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), artificial chromosome (PAC), sticking grain or F derived from P1- Sticking grain (fosmid).As this paper term used in the context being integrated into host cell gene group " nucleic acid carrier " refers to DNA from the nucleic acid carrier in the genome for being integrated into host cell.

Other examples of carrier include viral vectors, such as retroviral vector and slow virus carrier, in the application In it is especially interested.Slow virus carrier is such as widely used based on those of human immunodeficiency virus type 1 (HIV-1), because They can be integrated into non-proliferative cell.It is separated by the way that viral genome to be separated into the part of separation, such as by being placed in Plasmid on, viral vectors replication defective can be made.For example, by Salk Institute for Biological Studies The so-called first generation slow virus carrier of exploitation is built as being carried by packaging expression box, coating (envelope) expression cassette and transfer The three-plasmid system of body expression cassette composition." packaging plasmid " is containing completegag-polSequence, adjust (tatWithrev) and Auxiliary (vif、vpr、vpu、nef) sequence." envelope plasmid " holding blister under the control of cytomegalovirus (CMV) promoter Property stomatitis viral glycoprotein (VSVg) replace natural HIV-1 envelope protein.Third plasmid (" transferring plasmid " or " transfer vector ") Carry genetic modification, encapsulating sequence (ψ), Rev response element (RRE) sequence and CMV starting between long terminal repeats (LTR) Son is in host cell inner expression transgenosis.

Second generation slow virus carrier is characterized in that missing virulence sequencevpr、vif、vpuWithnef.Package carrier is reduced Extremelygag、pol、tatWithrevGene, therefore increase the safety of system.

In order to improve slow virus system, third generation carrier has passed through to be designed as follows: being removed from packaging constructtatGene And the LTR from carrier box is inactivated, therefore reduce the problem related to insertional mutagenesis effect.

Various slow virus are described in below with reference in document from generation to generation: the first generation: Naldini et al., (1996)Science 272(5259): 263-7；The second generation: Zufferey et al., (1997)Nat. Biotechnol.15(9): 871-5；The Three generations: Dull et al., (1998)J. Virol.72 (11): 8463-7, whole pass through reference and are hereby incorporated by reference in its entirety. Summary about slow virus carrier development is found in Sakuma et al. (2012)Biochem. J.443 (3): 603-18 and Pican o-Castro et al. (2008)Exp. Opin. Therap. Patents 18(5): 525–539。

Term " nonmammalian replication orgin " refers to the nucleic acid sequence for wherein causing and replicating and being originated from nonmammalian source Column.This enables nucleic acid carrier of the invention in suitable host cell (for example, microbial cell, such as bacterium or yeast Cell) in together with endogenous chromosome it is stable duplication and separate so that it can pass to host cell offspring (except when host is thin When born of the same parents are mammalian host cells).In mammalian host cell, the nucleic acid carrier with nonmammalian replication orgin It will be integrated into the endogenous chromosome of mammalian host cell or lost after mammalian host cell duplication.For example, tool There is artificial chromosome derived from the nucleic acid carrier such as bacterial artificial chromosome (BAC) of nonmammalian replication orgin, P1- (PAC), sticking grain or F sticking grain stable together with endogenous chromosome in bacterial cell (such as Escherichia coli) can replicate and divide From however, BAC, PAC, sticking grain or F sticking grain will be integrated or in mammal if introducing them into mammalian host cell It is lost after host cell duplication.Yeast artificial chromosome (YAC) can be stable multiple together with endogenous chromosome in yeast cells System and separation, however, YAC will be integrated or be answered in mammalian host cell if they are introduced into mammalian host cell It is lost after system.Therefore, in the present context, nucleic acid carrier of the invention serves as the storage cavern of DNA (that is, for generating reverse transcription disease Gene necessary to poison), it can be easily transferred in mammalian host cell to generate for generating retrovirus Stable cell lines.The example of nonmammalian replication orgin includes the replication orgin of bacterium, such as oriC, oriV or oriS, or The replication orgin of yeast, also referred to as autonomously replicating sequence (ARS element).

Nucleic acid carrier of the invention includes the replication orgin of nonmammalian and can accommodate at least 25 kilobase (kb) DNA.In one embodiment, nucleic acid carrier can accommodate at least 30,35,40,45,50,55,60,65,70,75,80, 85、90、95、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、 270, the DNA of 280,290,300,310,320,330,340 or 350kb.It should be understood that referring to that " carrying capacity " is logical with it Normal meaning, and imply that the upper limit of the Insert Fragment size of nucleic acid carrier (is not less than not less than claimed size The DNA of 25kb).

The purpose of the present invention is include base necessary to retrovirus is packed in single construct (i.e. nucleic acid carrier) Cause and amplifiable selectable marker gene.Therefore, nucleic acid carrier of the invention allows for accommodating big DNA Insert Fragment.To keep away Exempt from doubt, it should be understood that refer to that " nucleic acid carrier " or " artificial chromosome " does not refer to natural bacteria plasmid (for example, such as mesh The preceding plasmid used in transient transfection method) because these can not accommodate at least DNA of 25kb.Plasmid contains most Small insert is about 15kb significantly.Such nucleic acid carrier, which is also not, refers to biting for the maximum Insert Fragment for usually only accommodating 5-11kb Thallus.Therefore, in one embodiment, nucleic acid carrier of the invention is not plasmid, bacteriophage or episome.

Term " endogenous chromosome " (or host cell gene group) refers to such as thin in generation or introducing exogenous nucleic acid carrier The genome chromosome found in host cell before bacterium artificial chromosome.

Term " transfection (transfection) ", " conversion (transformation) " and " transduction as used herein (transduction) " can be used for describing by nonmammalian or the carrier of virus insertion target cell (i.e. host cell).Insertion Carrier is commonly referred to as the conversion of bacterial cell and the transfection of eukaryocyte, although insertion viral vectors is alternatively referred to as transduceed.Ability Field technique personnel will recognize usually used different non-viral transfection methods comprising but be not limited to using physical method (for example, electroporation, cell squeeze (cell squeezing), sonoporation, optics transfection, protoplast fusion, puncture transfection (impalefection), magnetic transfection, particle gun or partickle bombardment), chemical reagent is (for example, calcium phosphate, highly branched organic Compound or cationic polymer) or cation lipid (for example, lipofection).Many transfection methods need to keep Plasmid DNA molten Liquid is contacted with cell, then grow it and be directed to marker gene expression selected.

Term " promoter " refers to the sequence of driving gene expression.In order to drive high level expression, efficient promoter is used Such as non-retrovirus efficient promoter can be beneficial.The example of suitable promoter may include promoter such as people Cytomegalovirus (CMV) immediate early promoter, spleen focus-forming virus (SFFV) promoter, Rous sarcoma virus (RSV) are opened Mover or people's extension factor 1-α (pEF) promoter.

Tet operon (transcription activating of tetracycline control) can be used for can induce in the method for gene expression, wherein anti- Reversibly start or close transcription in the presence of one of raw element tetracycline or derivatives thereof (such as Doxycycline).In nature, Ptet promoter expresses TetR (aporepressor) and TetA (tetracycline antibiotic is pumped out to the albumen of cell).In the present invention, Tet operon may exist or be not present, for example, in one embodiment, Tet operon can reside in promoter.

Term " polyA signal " refers to polyadenylation signal sequence, such as is placed in the 3' of transgenosis, make host because The tail portion poly- adenosine (polyA) can be added to the end of newborn mRNA by son during transcription.The tail portion polyA is one section and is for up to The sequence of 300 adenosine ribonucleotides acid, protection mRNA from enzymatic degradation and additionally aid translation.Therefore, of the invention Nucleic acid carrier may include polyA signal sequence, such as people's beta globin or rabbit beta globin polyA signal, simian virus 40 (SV40) early stage or advanced stage polyA signal, actrapid monotard polyA signal or bovine growth hormone polyA signal.In an embodiment party In case, polyA signal sequence is people's beta globin polyA signal.

Term " intron sequences " refers to the nucleotide sequence removed by RNA montage from final gene product.It has shown Show the level for increasing gene expression using introne in the downstream in enhancers/promoters region and the upstream of cDNA Insert Fragment. The increase of expression depends on specific cDNA Insert Fragment.Therefore, nucleic acid carrier of the invention may include introne, such as people Beta globin introne, rabbit beta globin introne II or chimeric people beta globin-immunoglobulin introne.In an embodiment party In case, introne is people's beta globin introne and/or rabbit beta globin introne II.

Term " package cell line " refers to the thin of the gag and pol albumen and envelope glycoprotein gene for stablizing insertion Born of the same parents system.Alternatively, term " production cell line " refers to the stabilization between LTR sequence as described above including intended transgenic The package cell line of " transfer vector " component (used in such as transient transfection method) of insertion.It will be understood by those skilled in the art that Nucleic acid carrier as described herein can be used for generating package cell line (i.e. ought be at leastgag、polWithenvGene is present in nucleic acid carrier It is upper and when being incorporated to host cell) or production cell line (i.e. when nucleic acid carrier additionally comprise by withgag、polWithenvGene is together The transfer vector group timesharing being incorporated in host cell).

Term " stable transfection " be refer to pass to the reverse transcription virus gene of introducing they offspring it is (i.e. careful Born of the same parents) cell line, this is because transfection DNA have been incorporated into endogenous chromosome or via exogenous chromosome stablize heredity.

Term " expression construct " and " expression cassette " are used interchangeably, and as used herein, are to refer to drive one kind Or the functional expression unit of the expression of a variety of polynucleotides being incorporated to.Such box generally includes the polynucleotides and the multicore glycosides Component necessary to the transcription and translation of acid.For example, the box may include comprising promoter, translation initiation sequence signal, turn Record the nucleic acid sequence (i.e. recombinant DNA) of terminator (such as polyA sequence) and/or self cleavage peptide sequence (such as P2A sequence).In In one embodiment, the single expression box includes promoter and/or transcription terminator.In one embodiment, described Single expression box includes two genes separated by IRES, both from single promoter transcription.The construct can also contain There is any other additional component known to this field, can be enhanced or provide the bigger control to the transcription and translation of polynucleotides System.In one embodiment, the additional component can be any nucleic acid sequence from SV40 genome.

Nucleic acid carrier

According to an aspect of the invention, there is provided comprising nonmammalian replication orgin and at least 25 kilobase can be accommodated (kb) nucleic acid carrier of DNA, it is characterised in that the nucleic acid carrier includes to encode nucleic acid sequence below:

Gag and pol albumen,

Env albumen or its functional replacement, and

Amplifiable selected marker,

Wherein every kind of nucleic acid sequence is arranged as the individual expression construct in nucleic acid carrier.

The method for generating retroviral vector particle at present is related to transiently transfecting reverse transcription virus gene thin to host In born of the same parents.However, many disadvantages are related to this method, because its is at high cost, laborious and be difficult to scale up scale.A solution is The package cell line of engineered stable integration retrovirus packaging gene, to avoid the problem related to transient transfection.

It has been found by the present inventors that nucleic acid carrier as described herein can be used for generating retrovirus packaging or production cell System improves previously difficulty relevant to retroviral vector production method.For example, generating Retronituse encapsulated cell line Known method be included in introduce every kind of reverse transcription virus gene after carry out more wheel selections.The process can spend for up to six Month, and need a large amount of labour.After even obtaining package cell line after very long and laborious process, there is also incasing cells The report of viral vectors productivity being remarkably decreased during the suspension adaptation of system and expansion scale.

By by all reverse transcription virus genes include in nucleic acid carrier, then can be in single step by reverse transcription disease Virus gene is inserted into the endogenous chromosome of mammalian host cell.Therefore, as suggested herein, will be reduced using nucleic acid carrier Pressure is selected, quiet hour range is reduced and allows more rapidly to screen potential incasing cells.

In addition, the reverse transcription virus gene of nucleic acid carrier all will be integrated into mammalian hosts at term single gene seat In the endogenous chromosome of cell, risk that independent reverse transcription virus gene shrinks into oneself will be reduced and allow uniformly expression all Reverse transcription virus gene.

Integration at the single site in the genome of host cell allows to expand using amplifiable selectable marker gene The program of reverse transcription virus gene, wherein the amplification of reverse transcription virus gene is independent of reverse transcription virus gene and amplifiable choosing The internal connection of label is selected, if reverse transcription virus gene does not combine in term single gene seat, it is necessary to.

Therefore, by using nucleic acid carrier as described herein, it can choose the reverse transcription virus gene with amplification and lead Cause the improved packaging of the titre of retroviral vector particle or production cell.

In one embodiment, nucleic acid carrier additionally comprises the rna gene group of encoding hiv reverse transcriptase carrier granular Nucleic acid sequence.When transcribing the nucleic acid sequence, by by encapsidate in the retroviral vector particle that cell generates, and And therefore, the genome of retroviral vector particle is served as.It should be understood that the RNA of encoding hiv reverse transcriptase carrier granular The nucleic acid sequence of genome is generally included in " transfer vector " for transient transfection method.The metastasis transplanting physique grain is logical Often in two LTR sequences, ((it can be with by 3 ' LTR (its can be or can not be from inactivation (i.e. SIN) 3 '-LTR) and 5 ' LTR Include or can not include the area U5)) between containing with transgenosis (and optionally polyadenylation signal, and optionally further, For controlling/enhancing the nucleic acid sequence of the transcript and expression of transgenosis) promoter (such as SV40) that is operably connected and shell Body sequence (ψ).Therefore, in one embodiment, the rna gene group of the retroviral vector particle is included in two The one or more transgenosis encoded between LTR.

It in one embodiment, include the rna gene group of encoding hiv reverse transcriptase carrier granular in nucleic acid carrier Multiple copies of nucleic acid sequence (i.e. the component of transfer vector).It is expected that multiple copies of transfer vector lead to higher viral vectors Titre.For example, the nucleic acid sequence that nucleic acid carrier may include the rna gene group of encoding hiv reverse transcriptase carrier granular (shifts Carrier) two or more, such as three, four, five, six, seven, eight, nine or ten or more copy.

In one embodiment, nucleic acid carrier contains one or more recombination sites.This allows target sequence in recombinase In the presence of be integrated into the endogenous chromosome of mammalian host cell with site-specific fashion.Recombinase is recombinated at two Recombining reaction is catalyzed between site.

The site-specific recombination system of many types is known in the art, and can be used in the present invention any Suitable recombination system.For example, in one embodiment, recombination site is selected from or from λ bacteriophageint/attSystem, Cre/lox system, the FLP/FRT system of yeast, Gin/gix the recombination enzyme system, Cin recombinase of bacteriophage Mu of bacteriophage P1 System, Pin the recombination enzyme system and the R/RS system of pSR1 plasmid or any combination thereof of Escherichia coli.Further implement at one In scheme, recombination site isattSite (such as from λ bacteriophage), whereinattSite allows to determine in the presence of lambda integrase Point integration.It should be understood that refer to " lambda integrase " include refer to still withint/attThe mutant integrase of System compatible, such as The lambda integrase of the modification described in WO 2002/097059.

In one embodiment, nucleic acid carrier is selected from: bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), P1- derivative artificial chromosome (PAC), F sticking grain or sticking grain.In another embodiment, nucleic acid carrier is bacterium people Work chromosome (BAC).

Bacterial artificial chromosome

Term " bacterial artificial chromosome " or " BAC " refer to the DNA construct from bacterial plasmid, can accommodate the outer of large size Source DNA Insert Fragment.They can usually accommodate the maximum DNA Insert Fragment of approximate 350kb.BAC exploitation sufficiently characterizes certainly Bacterium functionality fertility plasmid (F- plasmid) rises containing promotion plasmid equally distributed distribution gene after bacterial cell division. This allows BAC to be replicated and separated by stablizing together with endogenous bacteria genome (such as Escherichia coli).BAC usually contains at least one A replication orgin copy is (such asoriSOroriVGene),repEGene (being adjusted for plasmid replication and copy number) and distribution base Because (such assopA、sopB、parA、parBAnd/orparC), ensure that BAC stablizing in bacterial cell maintains.BAC is day So cricoid and supercoil, making them than linear artificial chromosome, such as YAC is easier to restore.They also can be used Simple method such as electroporation relatively easily introduces bacterial host cell.

In one embodiment, bacterial artificial chromosome includesoriSGene.In one embodiment, bacteria artificial Chromosome includesrepEGene.In one embodiment, bacterial artificial chromosome includes distribution gene.It is further real at one It applies in scheme, distribution gene is selected fromsopA、sopB、parA、parBAnd/orparC.In one further embodiment, carefully Bacterium artificial chromosome includes sopA and sopB gene.

It can be obtained from commercial source for the BAC in the present invention, such as the pSMART BAC from LUCIGEN (right In complete skeleton sequence, referring to Genome accession number EU101022.1).The BAC contains L-arabinose " copy " system, ContainoriVMedium copy replication orgin, it is only just active in the presence of TrfA replication protein.The gene of TrfA can be in L- Arabinose inducible promoteraraC-P _BAD Control under be integrated into the genome of bacterial host cell (referring to Wild etc. People, (2002)Genome Res.12(9): 1434–1444).The expression of the addition induction TrfA of L-arabinose, TrfA are living Change oriV, causes plasmid replication to for up to 50 copy/cells.

Yeast artificial chromosome

Term " yeast artificial chromosome " or " YAC " refer to that wherein cerevisiae dna is integrated into the chromosome in bacterial plasmid.They contain There are autonomously replicating sequence (ARS) (i.e. replication orgin), centromere and telomere.Different from BAC, YAC is linear, and is therefore being contaminated Yeast telomere is contained in each end of colour solid, to protect end from degradation when it is transferred on host cell offspring.YAC can To accommodate a certain range of DNA Insert Fragment size；From any size of 100-2000kb.

Artificial chromosome derived from P1-

Term " artificial chromosome derived from P1- " or " PAC " refer to the DNA structure of the DNA from P1- bacteriophage and bacterium F- plasmid Build body.They can usually accommodate the maximum DNA Insert Fragment of approximate 100-300kb, and the clone being used as in Escherichia coli carries Body.PAC has the advantages that similar with BAC, such as is easy to purify and introduces bacterial host cell.

Sticking grain and F sticking grain

Term " sticking grain " refers to the DNA construct from bacterial plasmid, in addition contains from phageλcosSite.Sticking grain Usually contain bacterial origin of replication (such asoriV), selected marker, cloning site and at least onecosSite.Sticking grain usually may be used To receive the maximum DNA Insert Fragment of 40-45kb.Sticking grain has been displayed in terms of ehec infection cell than normal bacterial plasmid More effectively.Term " F sticking grain " refers to the nonmammalian nucleic acid carrier similar with sticking grain, in addition to it is based on bacterium F- plasmid.Tool For body, they allow to clone big DNA fragmentation using the replication orgin of F- plasmid and distribution mechanism.F sticking grain usually can be with Receive the maximum DNA Insert Fragment of 40kb.

Retrovirus

Retrovirus is the virus family containing pseudodiploid single stranded RNA genome.Their encoded reverse transcriptase enzymes, from RNA Genome generates DNA, is then inserted into host cell DNA.Invention as described herein can be used for generating replication defect type reverse Record vector particles.Retroviral vector particle of the invention can be selected from or be originated from any suitable retrovirus.

In one embodiment, retroviral vector particle is originated from or selected from slow virus, α-retrovirus, γ- Retrovirus or foamy retrovirus, such as slow virus or γ-retrovirus, especially slow virus.At one into one It walks in embodiment, retroviral vector particle is the slow virus selected from HIV-1, HIV-2, SIV, FIV, EIAV and Visna. Slow virus can infect in nondividing the cell of (i.e. static), this becomes them for the attractive inverse of gene therapy Transcription vector.In one further embodiment, retroviral vector particle be HIV-1 or be originated from HIV-1.One The genome structure of a little retrovirus can be found in the art.For example, the details about HIV-1 can be from NCBI Genbank (Genome accession number AF033819) is found.HIV-1 is one of the retrovirus being best understood by, and therefore often As retroviral vector.

Reverse transcription virus gene

The shared nucleic acid sequence of all retrovirus can be explained in greater detail as follows:

Long terminal repeats (LTR): the basic structure of reverse transcription virus gene group includes 5'-LTR and 3'-LTR, reverse transcription disease Poison generate needed for gene it is therebetween or in which.LTR is needed for retrovirus integration and transcription.They can also act as Promoter sequence controls the expression (that is, they are cis acting genes) of reverse transcription virus gene.LTR is named as by three The subprovince domain of U3, R, U5 form: U3 is originated from the unique sequence in the end 3' of RNA；R is originated from two duplicate sequences in end of RNA； And U5 is originated from the unique sequence in the end 5' of RNA.Therefore, in one embodiment, nucleic acid carrier additionally comprises 5'- and 3'- LTR.In a further embodiment, can lack the area U5 of 5'LTR and with the tail portion non-HIV-1 polyA replacement (referring to Hanawa et al., (2002)Mol. Ther. 5(3): 242-51)。

In order to solve and generate the viral relevant safety issue that can be replicated, the area U3 of missing 3'LTR is passed through In section come develop from inactivation (SIN) carrier, the area U3 includes the combination of TATA box and transcription factor Spl and NF- κ B Site is (referring to Miyoshi et al., (1998)J. Virol. 72(10): 8150-7).In reverse transcription and it is integrated in infected After in cell, which is transferred in 5'LTR, this leads to the transcriptional inactivation of LTR.This is referred to as based on the slow disease from inactivation The carrier system of poison, may include in the present invention.

ψ: it takes a turn for the worse by ψ (psi) sequence for being located at the end reverse transcription virus gene group 5' and records the shell of viral RNA Change.This field also it is known that, psi sequence downstream and extend refer togagSequence in code area is related to effective reverse transcription Viral vectors generates (referring to Cui et al., (1999)J. Virol. 73(7): 6171–6176).In one embodiment, Nucleic acid carrier additionally comprises Ψ (psi) sequence.

Primer binding site (PBS): reverse transcription virus gene group contains the existing PBS after the area U5 of 5'-LTR.It should TRNA primer needed for site combines starting reverse transcription.In one embodiment, nucleic acid carrier additionally comprises PBS sequence.

PPT: reverse transcription virus gene group contains referred to as polypurine area near the end 3' of reverse transcription virus gene group (PPT) purine of short extension.These PPT are functioned during reverse transcription as the RNA primer for the synthesis of positive chain DNA. Complicated retrovirus (such as HIV-1) contains second more centrally located PPT (that is, central polypurine area (cPPT)), It provides second site for originating DNA synthesis.The retroviral vector that coding cPPT has been displayed has turning for enhancing It leads with transgene expression (referring to Barry et al., (2001)Hum. Gene Ther.12(9): 1103-8).Implement at one In scheme, nucleic acid carrier additionally comprises 3'-PPT sequence and/or cPPT sequence.

The genome structure of above-mentioned noncoding region is well known to the skilled person.For example, about non-in HIV-1 The details of the genome structure of code area can be found from the Genome accession number AF033819 of NCBI Genbank, or for HIV-1 HXB2 (common HIV-1 Reference strains) is Genome accession number K03455.In one embodiment, noncoding region From the sequence at Genome accession number K03455, such as from base-pair 454-1126 (for R-U5-PBS-Gag), 7622- 8479 (for RRE) or 7769-8146 (for RRE), 4781-4898 (for cPPT), 9015-9120 and 9521-9719 are (right In dNEF-PPT-sinU3-R-U5).

Gag/pol:gagWithpolThe expression of gene depends ongagWithgagpolBetween translation move frame.Both exist The polyprotein cut during maturation.Primary structure matrix, capsid and the nucleocapsid protein of retroviral vector bygagIt compiles Code.polGene encoding hiv reverse transcriptase enzyme: i) reverse transcriptase, the reverse to retrovirus rna gene group to double-stranded DNA It records most important；Ii) integrase enables retrovirus DNA genome to be integrated into host cell chromosome；And Iii) protease cuts the polyprotein of synthesis to generate the maturation and functional protein of retrovirus.In an embodiment party In case, the retroviral nucleic acid sequence of encoding gag and pol albumen is originated from HIV-1 HXB2 sequence, is available from Genome and steps on Record K03455, such as base-pair 790-5105.

Env:envThe surface of (" coating ") gene encoding hiv reverse transcriptase coating and cross-film component (such as the sugared egg of HIV-1 White gp120 and gp41) and participate in retrovirus-cell membrane fusion.In order to widen the tissue tropism of retroviral vector, this Retroviral vector described in text can use the envelope protein pseudotyping from another virus.Pseudotyping, which refers to, passes through change Glycoprotein (GP) on retroviral vector particle from other enveloped virus (for example, by using obtaining or being originated from other packets The GP of film virus uses synthesis/artificial GP), the host cell range of retroviral vector (including slow virus carrier) can be with The process for being extended or being changed.Due to its extensive preferendum and high carrier granular stability, it to be used for pseudotyping retrovirus vector The most common glycoprotein of body is vesicular stomatitis virus GP (VSVg).However, it should be understood to the one skilled in the art that other glycoprotein can be with For pseudotyping (referring to Cronin etc., (2005)Curr. Gene Ther.5 (4): 387-398, by reference with it It is integrally incorporated herein).The selection of virus for pseudotyping may also depend upon the type of cell and/or organ to be targeted, because To have shown that some false types have organization type preference.In one embodiment, the function replacement object of env albumen is blister Property stomatitis viral glycoprotein.

In one embodiment, env albumen or its function replacement object is obtained from virus selected from the following or source: blister Tobamovirus (such as vesicular stomatitis virus), lyssavirus is (for example, hydrophobin, mokola virus (Mokola Virus)), arenavirus (for example, lymphocytic choriomeningitis virus (LCMV)), alphavirus is (for example, Ross river Virus (RRV), sindbis alphavirus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus), filamentous form virus (example Such as, the strain of Ebola virus Reston, zaire strain of Ebola virus, Lassa virus), Alpharetrovirus is (for example, the white blood of fowl Virus (ALV)), Betaretrovirus (for example, sheep pulmonary adenomatosis virus (JSRV)), Gammaretrovirus is not (for example, Lip river Buddhist nun murine leukemia virus (MLV), gibbon ape leukemia virus (GALV), cat endogenous retrovirus (RD114)), δ-is inverse Retroviral category (such as 1 type of human T-cell lymphotropic virus (HTLV-1)), Spumavirus (such as human bubble virus), slow disease Poison belongs to (such as Mei Di-Wei Sina is viral (MVV)), coronavirus genus (such as SARS-CoV), and Respirovirus is (for example, celestial Platform virus, respiratory syncytial virus (RSV) (RSV)), Hepacivirus (such as Hepatitis C Virus (HCV)), influenza virus (such as influenza A) and nuclear polyhedrosis virus (such as autographa california multicore polyhedrosis virus (AcMNPV)).In In one further embodiment, env albumen or its functional replacement obtained from or be originated from vesicular stomatitis virus.In the reality It applies in scheme, the albumen of vesicular stomatitis virus glycoprotein (VSVg) can be used, retroviral particle is felt It contaminates wider host cell range and eliminates the chance that recombination generates wild type envelope protein.In a further embodiment In, the retroviral nucleic acid sequence of coding env albumen or its functional replacement, which is originated from, is available from Genome accession number Sequence at J02428.1, such as base-pair 3071 to 4720.

Structural gene as described herein is all common for all retrovirus.Other auxiliary gene is found in not In the retrovirus of same type.For example, slow virus, such as HIV-1, are known as containing other six kindsrev、vif、vpu、vpr、nefWithtatAuxiliary gene.Other retrovirus can have an auxiliary gene similar with gene described herein, however it May be not always to be given and title identical in document.Bibliography such as Tomonaga and Mikami (1996)J. Gen. Virol.77 (Pt 8): 1611-1621 describe various retrovirus auxiliary genes.

Rev: auxiliary generev(" regulator of virion ") coding combines Rev response element (RRE) and promotes to reverse Record the auxilin of virus transcription object output.The protein product of the gene allows the reverse transcription disease containing Rev response element (RRE) Malicious mRNA segment is exported from nucleus into cytoplasm.Prediction RRE sequence is to form complicated foldable structure.This of rev is special The close coupling of effect reflection montage and core output step.In one embodiment, nucleic acid carrier includes RRE sequence.One In a further embodiment, RRE sequence is originated from HIV-1 HXB2 sequence, is available from Genome accession number K03455, such as Base-pair 7622 to 8479 or 7769 to 8146, especially base-pair 7622 to 8479.

Rev combination RRE and promote single montage (env、vif、vprWithvpu) or non-montage (gag、polAnd genome RNA) the output of virus transcription object, therefore cause downstream events such as gene translation and packaging (referring to Suhasini and Reddy (2009) Curr. HIV Res.7(1): 91-100).In one embodiment, nucleic acid carrier additionally comprises auxiliary generevOr it is similar to gene (that is, from other retrovirus or function similar system).IncludingrevGene ensures reverse transcription disease The RNA transcript of poisonous carrier genome is effectively exported to cytoplasm from nucleus, especially if RRE element be also included within to On the transcript of transhipment.In a further embodiment,rev(i.e. HIV-1 is cloned by gene and Genome accession number M11840 12 cDNA, HIVPCV12 locus) base-pair 970 to 1320 have at least 60% sequence identity, such as at least 70% sequence Identity.In an alternative embodiment,revGene and Genome accession number K03455.1 (i.e. human immunodeficiency virus 1 Type, HXB2) base-pair 5970 to 6040 and 8379 to 8653 have at least 60% sequence identity, such as at least 70%, 80%, 90% or 100% sequence identity.

Auxiliary gene is considered replicating in retrovirus and working in pathogenesis, therefore much current viruses carry Body production system does not include some in these genes.It is usually existingrevIt is an exception, or can be used and be similar torev/ The system of RRE system.Therefore, in one embodiment, coding auxiliary gene is destroyedvpr、vif、vpu、tatWithnefOr class Like one of auxiliary gene or a variety of nucleic acid sequences, so that RNA of the auxiliary gene from retroviral vector particle Encoding function auxilin is removed or is unable in genome.In a further embodiment, auxiliary gene is destroyedvpr、vif、vpu、tatWithnefOr in similar auxiliary gene at least two or more, it is three or more, four kinds or more Or all, so that the auxiliary gene is removed from the rna gene group of retroviral vector particle or to be unable to encoding function auxiliary Help albumen.Remove function auxiliary gene can not need to remove whole gene；It removes portion gene or destroys gene with regard to enough.

It should be understood that the nucleic acid sequence of coding Replication defective retroviral vector particle can be with retrovirus The wild type gene for the retrovirus that carrier granular is based on is identical or wild type gene from the retrovirus, that is, Sequence can be include in wild-type virus genetically or other modes change version sequence.Therefore, nucleic acid is introduced to carry Reverse transcription virus gene in body or host cell gene group can also refer to the codon optimization version of wild type gene.

Gene magnification

In nature, gene magnification is to characterize the process of some normal development states, such as Drosophila melanogaster (Drosophila melanogaster) in ovum occur, (Spralding et al. in the amplification of chorion gene especially in ovary (1980) PNAS, 77: 1096-1100).Gene magnification is also the frequent event in tumour cell, and wherein it is by causing cancer The enhancing of gene expression and (the Albertson D.G. (2006) that plays a major role in oncogene activationTrends Genet. 22: 447-455)。

" gene magnification " refers to such process, by the process, the specific dna sequence (i.e. gene) of genome relative to Other sequences in genome disproportionately replicate, so that the DNA sequence dna of amplification becomes than this out-of-proportion duplication The higher copy number of the copy number that initially there are in preceding genome exists.As " expanded herein in regard to used in gene or nucleic acid sequence Increase " or " amplification " refer to and copy the gene or nucleic acid that are present in host cell line by gene magnification with two or more Sequence.

The natural phenomena of gene magnification is developed as increase the recombination base that cell line generates in biological-pharmacy Because of the mode of the titre of product.In the case where in the genome that recombination has been integrated into host cell, then it can pass through choosing Select the cell line of recombination has been expanded after being wherein integrated into host cell gene group increase the copy number of recombination and With the amount of the recombinant protein of expression.

" amplifiable selectable marker gene " refers to that permission expands the gene under growth conditions appropriate as used herein Gene.On the contrary, term " selectable marker gene " as used herein and as described in more detail below, which refers to, not to be allowed to expand Selectable marker gene.The amplifiable selectable marker gene can increase expression product (that is, by amplifiable by expanding The expression of the albumen of selectable marker gene coding) and the shortage of inhibitor or essential metabolite is responded.When using inhibitor, When such as drug toxicity, the expression of the amplifiable selected marker increases the resistance for assigning and being directed to the inhibitor.Implement at one In scheme, the amplifiable selectable marker gene can be characterized as being can be complementary with auxotroph host.Implement at one In scheme, the amplifiable selected marker is under the control of promoter.In one embodiment, the promoter is that SV40 is opened Mover.

Term " selectable marker gene " as used herein refer to will be helpful to selection actively expression insertion gene (such as Nucleic acid carrier) cell but do not allow expand gene.The example of suitable selected marker includes the resistance encoded to antibiotic Enzyme (i.e. antibiotics resistance gene), such as kanamycins, neomycin, puromycin, hygromycin, blasticidin S or rich Lay be mould Element.Another example of suitable selected marker is fluorescin, such as green fluorescent protein (GFP), red fluorescent protein (RFP) or blue fluorescent protein (BFP).

" amplifiable selected marker " and " selectable marker " are referred respectively to by amplifiable selected marker base as used herein The product (that is, zymoprotein) of cause and selectable marker gene coding.

It can be by being expanded with amplifiable selectable marker gene stable transfection host cell come induced gene.Stable transfection Host cell is subjected to the drug toxicity of increasing concentration, it is known that the drug toxicity inhibits amplifiable selected marker.For example, can be Cultivate the cell of transfection in culture medium containing drug toxicity, the concentration of the drug toxicity realize by parental cell (i.e. not The cell of transfection) be plated in the culture medium containing drug toxicity after 3-5 days in greater than 98% cell kill.Alternatively, The cell of transfection can be sky at 37 DEG C in air in the culture medium containing drug toxicity (such as MTX or MSX) It is cultivated 14 to 21 days under shake in the wet atmosphere of 8% CO in gas, or until vigor > 90%.It, can be with by this inhibition Select cell colony, the expression with increased amplifiable selected marker, and therefore resisting to the drug of concentration used Property.Other method for induced gene amplification is known to the skilled in the art, such as OptiCHO Express examination Method described in agent box handbook (Life Technologies).

All expression constructs that nucleic acid carrier of the invention allows wherein to contain are single in host cell gene group It is integrated at locus.The amplification of amplifiable selectable marker gene also causes the amplification of surrounding DNA sequences.As a result, whole together The surplus DNA sequence for the nucleic acid carrier being bonded in host cell gene group will be also amplified.By this method, by using this paper institute The nucleic acid carrier stated can be provided for expanding retroviral vector gene of the stable integration into host cell gene group Method, independent of passing through the internal of a variety of retroviral vector genes of host cell and amplifiable selectable marker gene Connection integration.

Every kind of amplifiable selected marker has relevant selective agent (i.e. drug toxicity), during expanding with selection scheme It is added into cell culture medium.Different amplifiable selected markers/selective agent combination is known in the art, and in this hair Any suitable amplification and selection scheme can be used in bright.Suitable amplifiable selected marker/selective agent combination includes adenosine Deaminase/deoxycoformycin, aspartate carbamyl-transferase/N (phosphate base)-L-Aspartic acid, dihydrofoilic acid are also Protoenzyme/methotrexate (MTX), glutamine synthelase/methionine sulfoxide imines and metallothionein-l/ heavy metal.

In one embodiment, the amplifiable selected marker is dihyrofolate reductase (DHFR).DHFR selecting party Method is related todhfrThe expression construct of gene (that is, amplifiable selectable marker gene) is incorporated to nucleic acid carrier, thus induces Pressure is selected to the DHFR of other expression constructs of nucleic acid carrier.Host cell is transfected with nucleic acid carrier, and in increasing concentration DHFR inhibitor methotrexate (MTX) (MTX) in the presence of grow, with selection expanded the external source being integrated into host genomedhfrGene and the adjoint remaining cell for integrating nucleic acid carrier.

What it is for gene magnification includesdhfrThe expression construct of gene is well-known in the art, and for example describes In Subramani et al. (1981)Mol. Cell. Bio.2:854-864 and Walls et al. (1989)Gene 81: 139-149。

In one embodiment, describeddhfrGene and Genome Accession NM_010049.3 have at least 60% sequence Identity, such as at least 70%, 80%, 90% or 100% sequence identity.In one embodiment, DHFR and genome log in Number NP_034179 has at least 60% sequence identity, such as at least 70%, 80%, 90% or 100% sequence identity.

In one embodiment, the expression construct of the nucleic acid sequence comprising encoding D HFR can be further included from ape Component necessary to simian virus 40 subgenomic transcription and translation polynucleotides.

In one embodiment, it is originated from comprising encoding the expression construct of the nucleic acid sequence of amplifiable selected marker pSV2-dhfr, such as Subramani et al. (1981)Mol. Cell. Bio.Disclosed in 2:854-864.

In a further embodiment, the expression construct of the nucleic acid sequence comprising encoding amplifiable selected marker Nucleic acid sequence includes the nucleic acid sequence of SEQ ID NO:1.The nucleic acid sequence of SEQ ID NO:1 is as shown in Figure 2.

In another embodiment, the amplifiable selected marker is glutamine synthelase (GS).GS selection method Being related to willgsGene is incorporated to nucleic acid carrier, thus selects pressure to other expression constructs induction GS of nucleic acid carrier.Host is thin Born of the same parents are transfected with nucleic acid carrier, and are grown in the presence of the GS inhibitor methionine sulfoxide imines (MSX) of increasing concentration, with choosing It selects to have expanded and be integrated into host genomegsGene and the adjoint remaining cell for integrating nucleic acid carrier.

What it is for gene magnification includesgsThe expression construct of gene is well-known in the art, and is for example described in WO874462 and Bebbington et al. (1992)Nature 10:169-175)。

By using amplifiable selected marker and Correlation selection agent in this way, it can expand in host cell gene group and have There is the region of selection pressure, thus increases the copy number of amplifiable selected marker and nucleic acid carrier.Then, for retrovirus The titre of carrier production screens the cell line grown by the amplification and selection of such round, and best clone is selected to be used for Large-scale production.

In general, the concentration of amplification wheel number and the related reagent of use does not both set or fixes.Instead, it expands and selects Scheme usually becomes gradually stringent, until reaching the point of production threshold value.It has been observed that until close to the " steady of protein production Phase ", the protein production observed level it is usually directly proportional to the increase of gene copy number (Bebbington and Hentschel,DNA Cloning Volume III, IRL press, 1987)。

In a preferred embodiment, the host cell is negative for amplifiable selected marker.That is, The host cell does not include endogenous amplifiable selectable marker gene.For example, when using DHFR as amplifiable selected marker When, it is preferred to use host's strain of DHFR feminine gender, such as CHO DG44 or CHO DUX-B11.In one embodiment, the place Chief cell is host's strain of GS feminine gender.In one embodiment, it is endogenous to knock out to modify the host cellgsGene.It knocks out The method of the specific gene of cell is well-known in the art, for example, using Zinc finger nuclease.In a preferred embodiment In, the host cell is GS feminine gender HEK293T cell.However, the present invention is not only restricted to the selection of particular host cell system.It can To use any cell line in the method for the invention, with fast growth rate (for example, the doubling time is for 12 hours or more It is short) and the amplifiable selectable marker gene of external source can be expanded with reasonable rate without in the amplification of similar or higher rate The amplifiable selectable marker gene in source, so that the amplifiable selected marker of the external source may be used as dominant marker.

The copy number of the ability and amplifiable selected marker that are grown in the selective agent of increasing concentration by determining cell increases Add correlation, cell line that identification is transduceed with dominant marker (i.e. the amplifiable selected marker of external source) (this can by via Southern trace proves that the increase of the copy number of amplifiable marker directly measures or by via Northern trace or qPCR Prove that the increase of the amount of the mRNA generated by amplifiable marker measures indirectly).

In the case where host cell includes endogenous amplifiable selectable marker gene, other than amplifiable selected marker, Nucleic acid carrier can further include the nucleic acid sequence of encoding selection markers.The host cell is marked with comprising amplifiable selection The transfection of the nucleic acid carrier of note and selectable marker.Selectable marker assign antibiotic resistance in the case where, first against The host cell of the ability selection transfection grown in the presence of corresponding antibiotic such as bleomycin or hygromycin.With the party Formula, it is possible to select to have used the host cell of nucleic acid carrier Successful transfection before induced gene amplification.Then for gradually The ability selection cell grown in the selective agent of enrichment degree such as MTX or MSX.

Therefore, in one embodiment, the nucleic acid carrier additionally comprises selectable marker gene.Further at one In embodiment, selectable marker is antibiotics resistance gene, such as bleomycin, kanamycins or puromycin-resistant base Cause, especially bleomycin (ZeoR) resistant gene.In another further embodiment, the bleomycin resistance gene It is originated fromStreptoalloteichus hindustans bleGene, for example, see GenBank accession number X52869.1, from alkali Base is to 3 to 377.In another embodiment, the selectable marker is hygromix phosphotransferase.

Additional component

Nucleic acid carrier of the invention may include other additional component.It is, for example, possible to use these additional features for example to help Help stable transcript for translating, increasing gene expression dose and opening/closing genetic transcription.

The retroviral vector particle that the present invention generates can be used in the method for gene therapy.Therefore, implement at one In scheme, nucleic acid carrier additionally comprises one or more transgenosis.For example, when target gene does not have in mammalian host cell When correct expression, therefore the invulnerable release of target gene is introduced as transgenosis.In other words, which can be therapeutic activity base Cause, coding can be used for treating or improving the gene product of target disease.Transgenosis codified such as antisense RNA, ribozyme, albumen (such as tumor suppressor protein), toxin, antigen (it can be used for induction of antibodies or helper T lymphocyte or cytotoxic T cell) or Antibody (such as single-chain antibody).The transgenosis can encode expectation generated in cell in vitro, in vitro or in vivo it is any Polypeptide or RNA.In one embodiment, the transgenes encoding beta globin.Transgenosis can be from another cell type Or another species obtain, or are synthetically prepared.Alternatively, transgenosis can be obtained from host cell, but be operably coupled to Those different regulatory regions present in natural gene.Alternatively, transgenosis can be the gene being present in host cell not Iso-allele or variant.

It is expected that multiple copies of the transfer vector containing transgenosis lead to higher retroviral vector titre, therefore In one embodiment, nucleic acid carrier include transgenosis multiple copies, such as transgenosis two or more, especially three A or more copy.In some cases, it is desirable to which more than one gene product treats disease, therefore at one further In embodiment, nucleic acid carrier additionally comprises two or more, such as three or more or four kinds or more different Transgenosis.

The purpose of gene therapy is the inhereditary material of living cells to be modified for therapeutic purposes, and it is related to function base Because being inserted into cell to realize therapeutic effect.The retrovirus vector generated using nucleic acid carrier as described herein and host cell Body can be used for transfected target cells and induce the expression with the gene of potential treatment meaning.Therefore retroviral vector is available The mammalian body of the patient's condition (including but not limited to genetic disease, cancer and certain virus infections) is suffered from treatment, such as Human agent.

It will be understood by those skilled in the art that nucleic acid sequence can be operably associated with control sequence appropriate.For example, institute Stating nucleic acid sequence can be with expression control element, such as transcription/translation control signal, replication orgin, polyadenylation signal, interior Portion's ribosome entry site (IRES), promoter and/or enhancer etc. are operably associated.

In one embodiment, nucleic acid carrier additionally includes transcription regulatory element.For example, any element described herein It can be operably coupled to promoter, allow to control expression.The promoter being mentioned above may include known starting Son (complete or a part), can be it is continuously working or derivable, for example, regulatory protein there are the case where Under.In one embodiment, the promoter is viral promotors.In one embodiment, nucleic acid carrier additionally comprises Efficient promoter, such as CMV promoter.The promoter has the height for promoting the element encoded on nonmammalian nucleic acid carrier The advantages of horizontal expression.In another embodiment, CMV promoter includes the sequence from human cytomegalovirus strain AD169 Column.The sequence is available from Genome accession number X17403, such as base-pair 173731 to 174404.In an alternate embodiment In, the promoter is the SV40 late promoter from simian virus 40.The promoter being mentioned above may include known Promoter (complete or a part), can be it is continuously working or derivable, for example, the feelings existing for regulatory protein Under condition.

In one embodiment, promoter (such as CMV promoter) additionally comprises at least one Tet operon.Tet behaviour Vertical subsystem can be used for controlling the expression of contained retroviral sequence in nucleic acid carrier.In short, Tet aporepressor passes through knot The Tet operon site of introducing promoter is closed to block expression.Therefore, when Tet aporepressor is in conjunction with Tet operon, do not have There is gene expression.After addition tetracycline or Doxycycline, Tet aporepressor is isolated to allow promoter activity, therefore opens Open gene expression.Tet operon system be it is widely available, be such as available from the pcDNA of Invitrogen^TM4/TO mammal Tet operon used in expression vector.

In one embodiment, nucleic acid carrier additionally comprises tetracycline-resistance operon aporepressor (" Tet checks egg It is white " or " TetR ").In a further embodiment, Tet aporepressor is codon optimization.

In one embodiment, nucleic acid carrier additionally comprises insulator, such as chromatin insulator.Term " insulator " Refer to the gene order for blocking and interacting between promoter and enhancer.In a further embodiment, insulator is (all Such as chromatin insulator) it is present between each retroviral nucleic acid sequence (that is, individually between expression construct).This has Help prevent the promoter between adjacent retroviral nucleic acid sequence from interfering (that is, the promoter damage from a transcriptional units The expression of the adjacent transcriptional units of evil).Without being bound by theory, this, which is recognized as, helps to make to recombinate between retroviral sequence To generate the risk minimization for the virus that can be replicated.

It should be understood that if insulator is present in the nucleic acid carrier between each retroviral nucleic acid sequence, They can be used as the arrangement of the single expression construct in nucleic acid carrier.For example, each of encoding hiv reverse transcriptase nucleic acid sequence Sequence all has the promoter and/or introne and/or polyA signal of its own.

In one embodiment, chromatin insulator and jungle fowl (Gallus gallus) HS4 insulator sequence (such as Referring to Genome accession number U78775.2, base-pair 1 to 1205) at least 90% sequence identity, for example, at least 95% Sequence identity.

In one embodiment, nucleic acid carrier additionally comprises polyA signal.The use of polyA signal has protection MRNA is from enzymatic degradation and helps the advantages of translating.In a further embodiment, polyA signal obtained from or be originated from SV40, bovine growth hormone and/or people's beta globin.In one embodiment, polyA signal is originated from SV40 early stage polyA signal (for example, see Genome accession number EF579804.1, the base-pair 2668 to 2538 from minus strand).In one embodiment, PolyA signal be originated from people's beta globin polyA signal (for example, with reference to Genome accession number GU324922.1, base-pair 3394 to 4162)。

In one embodiment, nucleic acid carrier additionally comprises intron sequences.It is known in enhancers/promoters area downstream Increase the expression of Insert Fragment using introne with cDNA Insert Fragment (i.e. transgenosis) upstream.Further implement at one In scheme, intron sequences are people's beta globin introne or rabbit beta globin introne II sequence.In one embodiment, people Beta globin introne is originated from the sequence (such as from base-pair 476 to 1393) derived from Genome accession number KM504957.1.In In one embodiment, rabbit beta globin introne II is originated from the sequence derived from Genome accession number V00882.1 (for example, base To 718 to 1290).

In one embodiment, nucleic acid carrier additionally comprises groundhog hepatitis virus posttranscriptional regulatory element (WPRE). The presence of WPRE has shown that Enhanced expressing, and is therefore advantageously possible for obtaining high-caliber expression.Further implement at one In scheme, WPRE is originated from the sequence (for example, base-pair 1093 to 1684) derived from Genome accession number J04514.1.

In one embodiment, nucleic acid carrier additionally comprises internal ribosome entry site (IRES).IRES is structure The RNA element of change is typically found in the 5'- non-translational region (it is needed for assembly initiation complex) in 5'- cap downstream.IRES It is translated initiation factor to be identified, and allows the translation independent of cap.In a further embodiment, IRES is originated from brain Myocarditis virus (EMCV) genome (for example, with reference to Genome accession number KF836387.1, base-pair 151 to 724).

In one embodiment, nucleic acid carrier additionally comprises multiple cloning sites (MCS).MCS is containing multiple restricted DNA short-movie section in the nucleic acid carrier in site (such as 10,15 or 20 sites).These sites usually only go out in nucleic acid carrier It is now primary, to ensure that endonuclease is only cut at a site.This allows (to limit using suitable endonuclease Enzyme) it is readily inserted into reverse transcription virus gene.

It will be understood by those skilled in the art that construct can arrange in any order in nucleic acid carrier.In an example Property embodiment in, nucleic acid carrier includes following Insert Fragment: encode the nucleic acid sequence of amplifiable selected marker, encoding gag and The retroviral nucleic acid sequence of pol albumen encodes env albumen or the retrovirus of its functional replacement (such as VSVg) Nucleic acid sequence, encode auxiliary gene rev (such as rev sequence of codon optimization) or its similar to gene or function similar system Retroviral nucleic acid sequence, tetracycline-resistance operon aporepressor (TetR), internal ribosome entry site and selection mark Remember (such as bleomycin resistance selected marker) (that is, amplifiable selected marker-GagPol-Env-Rev-Tet aporepressor- IRES- antibiotic-resistance marker-remaining BAC sequence (" BAC skeleton ")；Or amplifiable selected marker GagPol- (wild type) VSVg- (codon optimization) Rev-Tet aporepressor-IRES- bleomycin resistance-pSMARTBAC).Further at one In embodiment, insulator (all such as chromatin insulators) be present in amplifiable selected marker,gagpol、envWithrevSequence Between respectively.In a further embodiment, promoter be present in amplifiable selected marker,gagpol、envWithrev Sequence respectively before.In one further embodiment, at least one copy of transfer vector sequence is (that is, include coding The nucleic acid sequence of the rna gene group of retroviral vector particle) it is present in front of amplifiable selected marker sequence.

In one embodiment, nucleic acid carrier includes following Insert Fragment: promoter (is such as optionally manipulated comprising Tet The CMV promoter of subsequence), introne (such as people's beta globin introne) encodes the nucleic acid sequence of amplifiable selected marker, PolyA signal (such as people beta globin polyA signal), insulator (such as chromatin insulator), promoter (such as optionally CMV promoter comprising Tet operon), introne (such as people's beta globin introne), the reverse of encoding gag and pol albumen Nucleic acid sequence is recorded, the retroviral nucleic acid of RRE, polyA signal (such as people beta globin polyA signal), insulation are encoded Sub (such as chromatin insulator), promoter (such as optionally including the CMV promoter of Tet operon sequence), introne is (all Such as people's beta globin introne), env albumen or the retroviral nucleic acid sequence (such as VSVg) of its functional replacement are encoded, PolyA signal (such as people's beta globin polyA signal), insulator (such as chromatin insulator), promoter (are such as optionally wrapped The CMV promoter of the operon sequence containing Tet), encode auxiliary gene rev or its similar to gene or functional similar system reverse Nucleic acid sequence, polyA signal (such as people's beta globin polyA signal) are recorded, insulator (such as chromatin insulator) opens Mover (such as CMV promoter), introne (such as rabbit beta globin introne), tetracycline-resistance operon aporepressor (TetR), internal ribosome entry site, selected marker (such as bleomycin resistance selected marker), polyA signal and Duo Ke Grand site.

Nucleic acid sequence can be successively introduced into nucleic acid carrier.This allows to be selected after each integration, to ensure Nucleic acid sequence in need is successfully integrated into nucleic acid carrier.Alternatively, will at least two or more nucleic acid sequences draw simultaneously Enter in nucleic acid carrier.

It should be understood that can be by standard molecule clone technology known in the art, such as using in restriction nuclease Additional genes as described herein are introduced into nucleic acid carrier by enzyme cutting and interconnection technique.Furthermore, it is possible to (such as, by standard technique Electroporation) nucleic acid carrier, especially BAC, PAC, F sticking grain and/or sticking grain introduced bacterial host cell, and (such as Escherichia coli are thin Born of the same parents, especially coli strain DH10B).

Purposes

A further aspect according to the present invention, provide for generate retrovirus packaging or production cell line as this Nucleic acid carrier defined in text.

Nucleic acid carrier described herein can be used for generating Retronituse encapsulated cell line, will greatly simplify reverse transcription disease The production of poisonous carrier.If should be understood that the nucleic acid sequence of the rna gene group (including transgenosis) of encoding hiv reverse transcriptase carrier Column are comprised on nucleic acid carrier, then this will be used to generate production cell line.

As described herein, stable retrovirus packaging or production cell line are developed to overcome and transiently transfect and reduce The relevant difficulty of retroviral vector particle titre will be useful.Nucleic acid carrier as described herein can be used for preparing institute Packaging or production cell line are stated, because they can accommodate the big DNA containing indispensable gene needed for retrovirus packaging Then the endogenous gene group of the gene integration to mammalian host cell can be neutralized in one step and be made by Insert Fragment It is expanded for unit, so that the amplification of required reverse transcription virus gene and then expression are directly proportional relative to each other.

Host cell

A further aspect according to the present invention, provides retrovirus incasing cell, it includes as single expression structure Build the coding nucleic acid sequence below of body:

Gag and pol albumen；

Env albumen or its functional replacement；With

Amplifiable selected marker；

Wherein the expression construct is all incorporated into one at the individual gene seat in retrovirus incasing cell genome It rises and is used as unit；And

Wherein the copy number of the unit is 2 or more.

It include that advantage on large-scale nucleic acid carrier is by all reverse transcription virus genes and amplifiable selected marker, it Can the preparation first in the microbial cell (such as bacterium or yeast cells), be physically easier to perform and manipulate, then in list It is integrated into mammalian cell in one step.Once reverse transcription virus gene is integrated into mammalian host cell, this With regard to mitigating selection pressure and reducing quiet hour section.The characteristic features of this method are, institute needed for creating package cell line There is reverse transcription virus gene that unit is all used as to be present in the term single gene seat of endogenous gene group, rather than is randomly dispersed within entire In endogenous gene group.This has the retrovirus incasing cell for generating and expressing all reverse transcription virus genes with same level Advantage, because they are all integrated in same gene seat, in contrast, the reverse transcription virus gene difference in previous known method And random integration, into the genome of entire host cell, this can lead to the expression of non-uniform level.

In addition, by with include to encode required retrovidae protein (gag, pol, env or its functional replacement) Nucleic acid sequence the identical nucleic acid carrier of all expression constructs on have amplifiable selected marker, together amplification expression building Body.

It should be understood that the unit can be at multiple and different positions in host cell gene group in different chromosomes It integrates more than once (although the nucleic acid sequence of all codings is all present in term single gene seat).Therefore, " term single gene is referred to Seat ", which is not excluded for nucleic acid sequence, to be packed/produces in retrovirus and be all incorporated into one at multiple locus in cellular genome A possibility that rising.This may be beneficial for the expression for increasing transgenosis, and may potentially improve reverse transcription disease Malicious titre.The copy number of the construct insertion between the cell colony for being originated from and individually cloning can be compared by qPCR.

In one embodiment, retrovirus incasing cell additionally comprises encoding hiv reverse transcriptase carrier granular The nucleic acid sequence of rna gene group.Such nucleic acid sequence can also be replaced with encoding gag and pol albumen, env albumen or its functionality Nucleic acid sequence and amplifiable selected marker for object are integrated in term single gene seat.

Therefore, a further aspect according to the present invention, provides retroviral producer cells, it includes as list The coding nucleic acid sequence below of only expression construct:

Gag and pol albumen；

Env albumen or its functional replacement；

Amplifiable selected marker；With

The rna gene group of retroviral vector particle；

Wherein the expression construct is all incorporated into one at the individual gene seat in retroviral producer cells genome It rises and is used as unit；And

Wherein the copy number of the unit is 2 or more.

In one embodiment, the retrovirus incasing cell is mammalian cell.It is further real at one It applies in scheme, it is thin that the mammalian cell is selected from 293 cell of HEK, Chinese hamster ovary celI, Jurkat cell, KS62 cell, PerC6 Born of the same parents, HeLa cell or derivatives thereof or functional equivalent.In one embodiment, the mammalian cell be CHO-K1 or CHO-K1SV.In another further embodiment, the mammalian host cell is 293 cell of HEK, or is originated from HEK 293 cells.Such cell can be attached cell system (i.e. they are to be attached to the monolayer growth on surface) or suspend and adapt to/non-patch Parietal cell system (i.e. they in the medium suspension growth).The cell of suspension growth is widely used in producing biological production in the industry Product produce because they grow high density and compared with attached cell and are easy expansion scale.In another further implementation In scheme, 293 cell of HEK is HEK 293T cell.Term " 293 cell of HEK " refers to commonly used in biotechnology Human embryo kidney 293 cells system.Specifically, HEK 293T cell is generally employed to produce various retroviral vectors.Suitably Other examples of commercially available cell line include T-REX (Life Technologies) cell line.Preferably, the mammal The not endogenous amplifiable selected marker of expression of cell.For example, in the case where amplifiable selected marker is DHFR, it is preferred to usedhfr ^- (dhfrIt is negative) cell line, such asdhfr ^- CHO cell line, or in the case where amplifiable selected marker is GS, it is excellent Choosing usesgs ^- (gsIt is negative) cell line, for example, modified withgsGene knockout is to generate the HEK293 of GS feminine gender HEK293T Cell line.The method for knocking out the specific gene of cell is well-known in the art, for example, using Zinc finger nuclease.

In a preferred embodiment, the host cell is negative for amplifiable selected marker.That is, The host cell gene group does not include endogenous amplifiable selectable marker gene.For example, when using DHFR as amplifiable choosing When selecting label, it is preferred to use host's strain of DHFR feminine gender, such as CHO DG44 or CHO DUX-B11.In one embodiment, The host cell is host's strain of GS feminine gender.In one embodiment, it is endogenous to knock out to modify the host cellgsBase Cause.In a preferred embodiment, the host cell is GS feminine gender HEK293T cell.However, the present invention is not only restricted to spy Determine the selection of host cell line.Any cell line can be used in the method for the invention, with fast growth rate (that is, Doubling time is 12 hours or shorter) and can be expanded with reasonable rate amplifiable selectable marker gene without with similar or Higher rate expands endogenous amplifiable selectable marker gene.Selective agent by discovery cell in increasing concentration (needs thin Compound of the amplifiable selected marker of cellular expression to survive) in grow ability and amplifiable selected marker copy number increase phase It closes, identifying has the energy that amplifiable selectable marker gene is expanded with the rate bigger than the rate for expanding endogenous selectable marker gene (this can directly be measured or be led to by the increase of the copy number via Southern trace proof amplifiable marker the cell line of power The increase for crossing the amount of the mRNA generated via Northern trace proof by amplifiable marker measures indirectly).

It should be understood that also can be applied to reverse of the invention above with respect to all embodiments of nucleic acid carrier description Record viral packaging/production cell.

Method

A further aspect according to the present invention provides the side for generating stable retrovirus packaging or production cell line Method comprising following steps:

(a) nucleic acid carrier as described herein is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian cell growth；With

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number.

Whether generation production or package cell line be respectively depending on nucleic acid carrier whether separately include or do not include encode it is inverse The nucleic acid sequence of the rna gene group of retroviral vector particles.

It, should if target gene is integrated into the endogenous chromosome with selected marker (such as antibiotics resistance gene) Method can further include selection wherein retroviral nucleic acid succeed integrate mammalian host cell.Therefore, at one In embodiment, the method includes selecting to have in culture be integrated into mammalian host cell after step a) Endogenous chromosome in carrier on the mammalian host cell of nucleic acid sequence that encodes.

The host cell of stable transfection is subjected to the selective agent (such as drug toxicity) of increasing concentration, it is known that the selective agent suppression Make amplifiable selected marker.For example, the cell of transfection, the drug toxicity can be cultivated in the culture medium containing drug toxicity Concentration realize in 3-5 days after parental cell (i.e. the cell of untransfected) is plated in the culture medium containing drug toxicity Greater than 98% cell kill.Alternatively, can be by the cell of transfection in the culture for containing drug toxicity (such as MTX or MSX) It is cultivated 14 to 21 days under shake in the wet atmosphere of aerial 8% CO at 37 DEG C in base, or until vigor > 90%. By this inhibition, cell colony can choose, the expression with increased amplifiable selected marker, and therefore to institute With the resistance of the drug of concentration.Step b) and c) can use gradually increase concentration selective agent repeat.In one embodiment, The amplification of foreign gene is by realizing the resistance for gradually increasing horizontal selective agent via repetition step b) and c) selection.

Method for induced gene amplification is known to the skilled in the art, such as OptiCHO Express examination Method described in agent box handbook (Life Technologies).

In one embodiment, it is thin to be selected from 293 cell of HEK, HEK 6E cell, CHO for the mammalian host cell Born of the same parents, Jurkat cell, KS62 cell, PerC6 cell, HeLa cell or derivatives thereof or functional equivalent.Further at one In embodiment, the mammalian host cell is 293 cell of HEK, or is originated from 293 cell of HEK.Such cell can be (i.e. they are being cultivated for attached cell system (i.e. they are to be attached to the monolayer growth on surface) or suspension adaptation/non-adherent cell system Suspension growth in base).In another further embodiment, 293 cell of HEK is HEK 293T cell or HEK 6E Cell.Other examples of suitable commercially available cell line include T-REX (Life Technologies) cell line.

In a preferred embodiment, the host cell is negative for amplifiable selected marker.That is, The host cell gene group does not include endogenous amplifiable selectable marker gene.For example, when using DHFR as amplifiable choosing When selecting label, it is preferred to use host's strain of DHFR feminine gender, such as CHO DG44 or CHO DUX-B11.In one embodiment, The host cell is host's strain of GS feminine gender.In one embodiment, it is endogenous to knock out to modify the host cellgsBase Cause.The method for knocking out the specific gene of cell is well-known in the art, for example, using Zinc finger nuclease.Preferably at one In embodiment, the host cell is GS feminine gender HEK293T cell.

However, the present invention is not only restricted to the selection of particular host cell system.It can be in the method for the invention using any Cell line can be expanded with fast growth rate (that is, the doubling time is 12 hours or shorter) and with reasonable rate Amplifiable selectable marker gene is without expanding endogenous amplifiable selectable marker gene with similar or higher rate.It is thin by finding The ability that born of the same parents grow in the selective agent (compound for needing cell to express amplifiable selected marker to survive) of increasing concentration Related to the increase of the copy number of amplifiable selected marker, identifying has with bigger than the rate for expanding endogenous selectable marker gene Rate expand the ability of amplifiable selectable marker gene cell line (this can by via Southern trace prove it is amplifiable The increase of the copy number of label directly measurement or the mRNA by proving to be generated by amplifiable marker via Northern trace The increase of amount measures indirectly).

Technical staff, which will realize, can be used appropriate method known in the art, such as the transfection, micro- that lipid mediates Injection, cell (such as microcell) fusion, electroporation or microparticle bombardment, introduce host cell for nucleic acid carrier.In a reality It applies in scheme, nucleic acid carrier is introduced by host cell by electroporation.It should be understood that be used to introduce the method for nucleic acid carrier Selection can be selected according to the type of the mammalian host cell used.

Once nucleic acid carrier will be by the endogenous of random integration to mammalian host cell in mammalian host cell In genome.Therefore, this method further includes the mammal place that selection has wherein been incorporated into the nucleic acid encoded on nucleic acid carrier Chief cell (for example, using antibiotic resistance selected marker, such as bleomycin resistance is marked).

It will be appreciated by persons skilled in the art that promoting the method for nucleic acid carrier integration, for example, if nucleic acid carrier is natural Cricoid (for example, BAC, PAC, sticking grain or F sticking grain), then linearized.Nucleic acid carrier can additionally comprise and mammal The endogenous chromosome of host cell shares the region of homology, to guide the selected site being integrated into endogenous gene group.In addition, If recombination site is present on nucleic acid carrier, these can be used for targeting recombination.For example, nucleic acid carrier can containloxPPosition Point allows targeted integration when combining with Cre recombinase (even if being used to the Cre/lox system from P1 bacteriophage).Or (or In addition), recombination site isattSite (such as from λ bacteriophage), whereinattSite allows to be determined in the presence of lambda integrase Point integration.This will allow reverse transcription virus gene targeted to the locus in endogenous gene group, allows high expression and/or stablizes Expression.

Other targeted integration methods are well-known in the art.It is, for example, possible to use the targetings of induced gene group DNA to cut Targeting recombination at chromosomal loci of the method cut to promote selection.These methods are usually directed to is cut using engineered System is cut to induce double-strand break (DSB) in endogenous gene group or notch by natural process (such as nonhomologous end Engage (NHEJ)) repair fracture or using recovery template reparation (that is, homology orientation is repaired or HDR).

By using such as engineered Zinc finger nuclease of specific nuclease (ZFN), transcriptional activators such as effect Object nuclease (TALEN) is referred to using CRISPR/Cas9 system and engineered crRNA/tracr RNA (' single guidance RNA') Lead specificity cutting and/or using based on Argonaute system nuclease (such as from thermus thermophilus (T. thermophilus), referred to as ' TtAgo', referring to Swarts et al., (2014)Nature507 (7491): 258-261), it can It cuts.The process for being used for HDR or NHEJ mediation is cut by core using targeting for one of these nucleic acid enzyme systems Acid is inserted into specific target position.Therefore, in one embodiment, also comprise will be using at least one nuclease for this method The nucleic acid sequence encoded on nucleic acid carrier is integrated into the genome (i.e. endogenous chromosome) of mammalian host cell, wherein institute The genome for stating at least one nuclease cutting mammalian host cell, so that nucleic acid sequence is integrated into the genome of cell In.In a further embodiment, at least one nuclease is selected from Zinc finger nuclease (ZFN), TALE nuclease (TALEN), CRISPR/Cas nucleic acid enzyme system and combinations thereof.

A further aspect according to the present invention, provides the retrovirus packet obtained by method defined herein Dress or production cell.

It can be used for generating using the cell line that method defined herein obtains and there is the retroviral vector for improving titre The cell line of particle.

It is a effective amount of reverse transcription disease for referring to transduction target cell such as Patient cells reference is made to term " titre " Poisonous carrier or particle.In one embodiment, high titre is, more than 10⁶TU/ml is without concentration (TU=transduced unit).

A further aspect according to the present invention provides the side for generating Replication defective retroviral vector particle Method comprising following steps:

(a) nucleic acid carrier as described herein is transfected into the culture of mammalian host cell；

(b) grow the mammalian host cell of transfection in the culture medium of the selective agent containing such concentration, the concentration The selective agent amplifiable selected marker that inhibits expression insufficient horizontal transfection mammalian host cell growth；

(c) mammalian host cell for the transfection that selection can be grown in the medium, wherein the food in one's mouth of the transfection selected Newborn animal host cell contains the nucleic acid carrier in the mammalian host cell genome for being integrated into transfection of amplification copy number； With

(d) food in one's mouth selected in further incubation step (c) under conditions of generating Replication defective retroviral vector particle Newborn animal host cell.

Step b) and c) can use gradually increase concentration selective agent repeat.Therefore, in one embodiment, external source base The amplification of cause is by realizing the resistance for gradually increasing horizontal selective agent via repetition step b) and c) selection.

It, should if target gene is integrated into the endogenous chromosome with selected marker (such as antibiotics resistance gene) Method can further include selection wherein retroviral nucleic acid succeed integrate mammalian host cell.Therefore, at one In embodiment, the method includes selecting to have in culture be integrated into mammalian host cell after step a) Endogenous chromosome in carrier on the mammalian host cell of nucleic acid sequence that encodes.

As it was noted above, in one embodiment, it is thin that the mammalian host cell is selected from 293 cell of HEK, CHO Born of the same parents, Jurkat cell, KS62 cell, PerC6 cell, HeLa cell or derivatives thereof or functional equivalent.Further at one In embodiment, the mammalian host cell is 293 cell of HEK, or is originated from 293 cell of HEK.Such cell can be (i.e. they are being cultivated for attached cell system (i.e. they are to be attached to the monolayer growth on surface) or suspension adaptation/non-adherent cell system Suspension growth in base).In another further embodiment, 293 cell of HEK is HEK 293T cell.Suitable city Other examples for selling cell line include T-REX (Life Technologies) cell line.

In a preferred embodiment, the host cell is negative for amplifiable selected marker.That is, The host cell gene group does not include endogenous amplifiable selectable marker gene.For example, when using DHFR as amplifiable choosing When selecting label, it is preferred to use host's strain of DHFR feminine gender, such as CHO DG44 or CHO DUX-B11.In one embodiment, The host cell is host's strain of GS feminine gender.In one embodiment, it is endogenous to knock out to modify the host cellgsBase Cause.The method for knocking out the specific gene of cell is well-known in the art, for example, using Zinc finger nuclease.Preferably at one In embodiment, the host cell is GS feminine gender HEK293T cell.However, the present invention is not only restricted to particular host cell system Selection.

It will be understood by those skilled in the art that condition used in method described herein will be thin depending on used host Born of the same parents.Typical condition, such as culture medium ready for use or temperature, are well-known in the art.In one embodiment, it trains It supports and is carried out by being incubated for mammalian host cell in a wetted condition.In a further embodiment, wet condition It is included in 37 DEG C, 5% CO₂The lower cell for being incubated for transfection.In one embodiment, it is trained using culture medium selected from the following It supports: the EagleShi culture medium (DMEM) that the DulbeccoShi containing 10% (vol/vol) fetal calf serum (FBS) is improved, serum-free UltraCULTURE^TMCulture medium (Lonza, catalog number (Cat.No.) 12-725F), BalanCD HEK293^®Culture medium (Irvine Scientific, catalog number (Cat.No.) 911615) or FreeStyle^TMExpress culture medium (Thermo fisher, catalog number (Cat.No.) 12338- 018)。

Cultural method appropriate is well known to the skilled person.For example, can be in suspension and/or without animal Cell is cultivated under conditions of component.In one embodiment, the cell is suitable in the culture medium of any volume, from 10 Culture in ml (such as in shaking flask) to 10 L, 50 L, 100 L or more (such as in the bioreactor).

In one embodiment, this method also comprises separation Replication defective retroviral vector particle.For example, In one embodiment, it is separated by using filter.In a further embodiment, filter is low egg White combination film (for example, 0.22 μm of low protein binding film or 0.45 μm of low protein binding film), such as Kynoar (PVDF) or Polyether sulfone (PES) artificial membrane.

Once the retroviral nucleic acid being present on nucleic acid carrier can be integrated into interior in mammalian host cell In random, term single gene seat in the genome of source.Integration step can promote as described above, for example, using linearisation and/or The region of shared homology.Recombination site can also be used for targeting recombination.

Once separation, retroviral vector particle can be concentrated for vivo applications.Method for concentration includes for example exceeding the speed limit Centrifugation, precipitating or anion-exchange chromatography.Ultracentrifugation is to have as the fast method that small-scale retroviral vector is concentrated .Alternatively, anion-exchange chromatography (such as using Mustang Q anion exchange bellows) or precipitating (such as use PEG 6000) particularly useful for handling the slow virus carrier supernatant of large volume.

A further aspect according to the present invention, it is inverse to provide the replication defect type obtained by method defined herein Retroviral vector particles.

Referring now to following non-limiting embodiment, the present invention will be described in more detail.

Embodiment