JP2002530070A - A sensitive screening system for envelope-defective recombinant viruses. - Google Patents

Abstract

  (57) [Summary] An indicator cell line that complements the envelope-deficient recombinant virus with trans can be used to amplify the virus. The present invention relates to a method for amplifying a retrovirus, for example, by exposing an envelope-defective retrovirus to cells containing a virus envelope gene, wherein the virus envelope encoded by the gene complements the retrovirus. . Furthermore, the method of the present invention relates to a method wherein the viral envelope is expressed on the surface of a virus particle produced by a cell. The method of the invention also relates to a method wherein the viral envelope is expressed by a cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION A sensitive screening system identifies envelope-defective recombinant viruses that occur during the production of lentiviral or retroviral vectors.

BACKGROUND OF THE INVENTION Generally, recombinant viruses are replication defective. However, such recombinant viruses can still be detrimental to vector production in some ways. First,
Recombinant virus can be propagated in vector producer cells. Second, recombinant viruses can interfere with vector transduction by competing during encapsidation of the viral particles. In addition, recombinant viruses can be harmful to vector recipients due to transfer of the vector's packaging function. It can cause a toxic or immune response in the transduced cells and host. Finally, the risk of further recombination events that can lead to the production of replication competent retroviruses can also be increased.

[0003] Such a recombinant virus may be derived from the recombination of two or more constructs used to produce the vector, or from one such construct and the endogenous vector expressed in the vector producer or target cells. It can result from a sex retroviral sequence. Recombinants generally contain the viral cis-acting sequences required for encapsidation and are transferred to target cells. Recombinants also generally include the retroviral or lentiviral gag / pol gene sequences.

[0004] The extent to which a defective recombinant (eg, an envelope-defective recombinant) contaminates a batch of vectors produced for clinical use or affects the performance of a vector producer system. , Often unknown. The risk of deletion versus replication-competent recombinant development depends on the construct encoding the envelope gene and gag
Due to the lack of any duplication between the construct encoding the / pol gene and the new split-genome packaging cell line for the retroviral vector, and the vector pseudotype
ng). Although it implies the generation of a lower risk replication competent recombinant, recombination between the gag / pol construct and the transfer vector carrying the foreign gene of interest has not yet been recognized in conventional screening. Envelope-deficient recombinants can and will be produced.

[0005] Lots of defective recombinantly contaminated vectors used in clinical trials can also be responsible for false positive results in certain assays used to monitor replication competent recombinants in recipients. .

[0006] Thus, sensitive detection and early elimination of defective recombinants is necessary to confirm and maintain the performance of the vector producer system, and to demonstrate the purity and safety of a batch of vectors. Is important. However, conventional assays used to monitor retroviral recombinants based on amplification through replication in an indicator cell line are defective, since defective recombinants are also replication defective. The body cannot be detected.

SUMMARY OF THE INVENTION [0007] The present invention provides transcomplementation in an indicator cell line that provides a lack of packaging function (eg, an envelope gene that detects envelope-deficient recombinants).
An amplification method for detecting a replication-defective recombinant based on transcomplementation is described. An important feature that complements the envelope is that it does not interfere with superinfection of the indicator cells, thereby allowing the amplification by pseudoreplication of the recombinant in a homogeneous culture under certain circumstances. rare.

DETAILED DESCRIPTION OF THE INVENTION Detection of replication-defective retroviral recombinants, particularly lentivirus-based vectors, provides a complementation function in trans for a complementation function suspected of being defective in the recombinant. It depends. For example, if detection of an envelope-deficient recombinant is desired, means for providing an envelope protein are utilized. It can be achieved by developing a transcomplementing cell line that expresses one or more components required to produce the virus particle, such as an envelope protein.

[0009] Indicator cell lines that express trans-complementing proteins (eg, envelope proteins, tat proteins, rev proteins or combinations thereof) can be constructed and used as screening agents in the present invention.

[0010] Preferably, the indicator cell line is a stable transformed cell expressing one or more transcomplementing factors. Generally, a stable transformed cell is one in which a transgene encoding the desired factor has been integrated into the host genome. It can be obtained, for example, by transfecting a vector carrying the transgene and known to be integrated into the host genome (eg, a retroviral vector, a transposon-based vector or an adeno-associated virus vector).
Or by using it.

[0011] Furthermore, since foreign gene products (eg, envelope proteins) can be toxic to host cells, it is desirable to regulate the expression of this transgene. For example, an inducible promoter can be used to control the expression of the transgene. Such inducible promoters are known in the art.

[0012] Otherwise, the production and maintenance of the transformed cells and the vector of interest employs materials readily available to those of skill in the art, as is known in the art. Any of a variety of host cells can be used. In addition, vectors and transgenes are known, and those skilled in the art can rely on known methods to construct a vector of interest.

Basically, any known vector carrying the trans-complementing gene of interest and any suitable host cell can be used. Known inducible regulatory systems can be used to regulate the expression of trans-complementing genes. Also, any known method for detecting expression of a virus or gene product that produces a defective recombinant (eg,
immunoassay for gag protein) can be used.

[0014] 29 expressing envelope protein G of vesicular stomatitis virus (VSV)
3G cell line (Ory et al., Proc. Natl. Acad. Sci. 93: 114).
00-11406, 1996) can be used to detect envelope-deficient viruses, where their entry into target cells can be mediated by the protein G. This G protein is interesting. This is because the envelope glycoprotein has been found to complement a wide range of viruses.
Expression of the VSV G protein in 293G is controlled in a tetracycline regulated manner.

The VSV G cell line, for example, nevertheless has the gag / pol
It can be used to amplify minimal amounts of envelope-deficient recombinant virus that can express and transfer genes. In the tetracycline regulatory system, these cells are maintained in the presence of tetracycline, which suppresses VSVG expression. 29
Removal of tetracycline from the culture medium maintaining 3G cells results in induction of VSV G protein on the surface of the indicator cells, thereby allowing efficient pseudotyping and / or entry of released virus particles. enable. The particles can then superinfective indicator cells that lead to amplification of the input viral recombinant.

The VSV G envelope is particularly useful by conferring very high infectivity on the virions, thereby increasing the sensitivity and rigor of the assay.

[0017] Amplification of the virus has been observed not only with viruses that carry the envelope glycoprotein, but also with viruses that lack or do not express the envelope glycoprotein. Thus, in the case of the VSV G protein, expression of the G protein on the surface of the target cell is sufficient to mediate productive infection independent of expression at the viral membrane.

In their view, the present invention relates to methods by which screening of vector recipients after treatment may be adapted. This method can also be used to amplify other enveloped viruses for which natural cell receptors must still be identified.

The assays of the invention find use, for example, in the production of efficient and safe HIV-based lentiviral vectors. Propagation of recombinant particles can be performed, for example, using HIV.
by immunoenzymatic assay to detect p24 gag antigen or by HIV
It can be detected by an RNA-PCR assay to detect the gag gene.
The presence of the defective recombinant can then be monitored by use of the indicator cells of the invention.

The methods of the present invention may also include other retroviruses, such as lentiviruses, such as various SIVs, FIVs, HIV-2, visna-maediviruses, goat arthritis-encephalitis virus, BIV and equine infectious anemia Virus), as well as other retroviruses (eg, spumavirus, mouse leukemia virus and mouse sarcoma virus), other mammalian type C viruses (eg, FeLV and simian sarcoma virus), HERV, type B virus (eg, mouse mammary tumor virus) ), Type D virus, HTLV, bovine leukemia virus and avian leukemia-sarcoma virus (e.g.,
Rous sarcoma virus and chicken myeloblastosis virus) and can be used to identify partially recombinant viruses that express and transfer the gag / pol gene. The recombinant particle detection system is tailored to the gene of the selected virus.

In another embodiment of the invention, the indicator cell line complements in trans one or both of the tat and rev regulatory genes essential for lentiviruses, in addition to the envelope gene. Such systems are useful for identifying partial recombinants that contain the lentiviral gag / pol genes but do not express those genes efficiently. This is because, for example, the regulatory sequences required for efficient expression of the gag / pol gene are missing or defective. Suitable assays are those that detect the expression of gag or pol.

In detecting envelope-deficient recombinants, the amplification provided by the present invention provides a significantly more efficient virus entry mediated by a complementary envelope protein when compared to the homologous or parental gene product. Arising from Thus, another use of the method of the invention is the rapid selection of viral genetic variants of a desired phenotype (eg, drug resistance or growth advantage). This selection can be performed without having to actually produce the infectious viral construct. This is because the complementary envelope protein can be produced by a cell (eg, an indicator cell of interest).

The present invention is now illustrated in the following non-limiting examples.

EXAMPLES For verification of the detection system, envelope-deficient recombinants were constructed by molecular cloning using known techniques. VSV G protein expression construct p
MD. G (which does not contain the HIV sequence) was used (Naldini et al., S
science 272: 263-267, 1996a). Envelope-deficient recombinant constructs and VSV. Viral particles were generated by co-transfecting the G expression construct into 293T cells (Naldini et al., 1996a
Proc. Natl. Acad. Sci. 93: 11382-1138
8, 1996b). This recombinant construct has an H containing all but the envelope sequence.
It was an IV-based vector.

The control virus was cloned with an envelope-deficient recombinant plasmid R8.7 de
IE and VSV. G-expressing plasmid pMD G (Naldini et al., 199)
6a, supra) was produced by means of transient transfection into 293T cells. The R8.7 plasmid was replaced with the plasmid pCMVDR8.74 (Dull
J. et al. Virol. 72: 8463-8471, 1998).
The oI fragment, which contains the HIV gag, pol, tat and rev genes but no accessory genes, was ligated with R8 (Gallay et al., Cell 83).
: 569-576, 1995).

Cells were seeded in 10 cm dishes 24 hours prior to infection and washed 2 hours before transfection. Add 14 culture media (IMDM, 10% FCS)
The time was changed and the transfected conditioned medium was replaced with 36 days of transfection.
Collected after hours. The conditioned medium was clarified by low speed centrifugation (1500 g) and passed through a 0.45 μm filter. The amount of virus particles in this medium was determined using an immunocapture assay (D) for HIV-1 p24 gag antigen.
uPont).

The VSV. The titration of virus particles on G indicator cells allowed the assessment of the sensitivity of this assay and provided a means to determine the amount of envelope-deficient recombinants in vector preparations.

Indicator 293G cells (Ory et al., Supra) and control 293 cells were seeded in 6-well plates at approximately 30% confluence 24 hours prior to infection. Immediately before infection, the cells were washed with fresh medium. The control virus was serially diluted 10-fold in medium without tetracycline, and 1 ml of each dilution was added to each well. Culture medium from infected cells,
Exchange regularly and amplification of recombinants was monitored by measuring p24 antigen in the supernatant. Infected cells were reduced to 1/5 after reaching confluence.
Divided into

As provided in FIG. 2, this system is a virus that capsidizes the envelope-deficient construct at a level of less than 20 fg of p24 equivalent (the minimum dilution used) during a 15-day incubation period. The inoculum of the particles can be detected. p
A gradual increase in the 24 antigen concentration was observed in the culture supernatant at the indicated number of days after infection. No amplification of the recombinant was seen when 293 cells lacking the VSV envelope were infected with the same amount of virus particles.

The data in FIG. 3 demonstrates the ability of 293G cells to support viral amplification after initial infection by a virus lacking envelope glycoprotein. Virions, e
pMD.nV encoding the nv gene and carrier DNA or VSV G. Produced by transient co-transfection of a plasmid encoding an HIV derivative containing any deletion of the G plasmid. Virions were normalized for p24 content and serially diluted (20 ng / ml, 0.5 ng / ml and 0.125 n
g / ml) and incubated with either induced 293 / G cells or control 293 cells. The expression of G in the 293G / cell line is
As regulated by tetracycline, these cells were maintained in the absence of tetracycline 24 hours prior to infection.

During the two-week period, infected cell supernatants were monitored for p24 content as a measure of virus amplification. The data in FIG. 3 are the results observed at the end of the two week period.

No p24 was detected in the supernatant of 293 cells incubated with envelope-deficient virions.

As expected, 293 cells infected with the VSV G pseudotyped virus produced low levels of p24, proportional to the viral input. The 293 cells did not expand throughout the incubation period. On the other hand, 293G cells produced increasing amounts of p24 at the same kinetics, whether or not pseudotyped or enveloped viruses were used for the initial infection.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows that the envelope-deficient recombinant is a packaging plasmid and HIV.
Figure 4 shows how they can be made from transfer vectors carrying sequences. The upper figure shows wild-type H
4 shows the IV-1 genome. In this figure, LTR is a long terminal repeat; SD is a splice donor site; GAG is a group antigen coding sequence; PRO is a protease coding sequence; POL is a polymerase coding sequence;
VIF and NEF are accessory molecules; CMV is a cytomegalovirus enhancer / promoter; poly A is a polyadenylation site;
The name V indicates the deletion of the envelope coding sequence; SA is the splice acceptor site; prom is the promoter; the transgene is the foreign gene of interest; Ψ is the encapsidation signal sequence. Yes; TAT and REV
Is a regulatory gene; and RRE is a Rev responsive element. The black box indicates the open reading frame of HIV, ie, a functional gene, in the three reading frames.

FIG. 2 shows the results of an assay aimed at determining the sensitivity of the method of interest.

FIG. 3 shows the results of virus amplification in the absence or presence of the VSV G envelope.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 Inventor Nardini, Luigi United States of America 94404, Foster City, Lakeside Drive 342, Cell Genesis, Inc. F-term (reference) 4B024 AA20 BA80 CA03 EA02 HA20 4B065 AA95Y AA97Y AB10 CA60

Claims (3)

[Claims] 1. A method for amplifying a retrovirus by exposing an envelope-deficient retrovirus to a cell containing a virus envelope gene, wherein the virus envelope encoded by the gene complements the retrovirus. Method. 2. The method of claim 1, wherein said viral envelope is expressed on the surface of virus particles produced by said cells. 3. The method according to claim 2, wherein the viral envelope is expressed by the cell.
The method of claim 1.