CZ20001483A3 - Suspension cellular lines for wrapping retroviral vectors - Google Patents

Abstract

The solution relates to the line of enveloping cells fully growing a suspension that is transfected with a retroviral plasmid or transreduced by a retroviral vector as well as procedures preparing such cell lines from suspension cell lines cell lines or adherent cell lines.

Description

Suspension cell lines for enveloping retroviral vectors

Technical field

The invention relates to a cell line of enveloping cells growing completely in suspension, which is transfected with a retroviral plasmid or transduced with a retroviral vector, and to a method of preparing such cell lines from suspension cell lines or adherently growing cell lines.

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides coat cell suspension lines for retroviral vectors and their use in the preparation of infectious replovable retroviral vectors.

Viruses consist both of the genome encoding the viral genes and of the viral capsid which, as in the case of retroviruses, can be surrounded by a membrane envelope.

Retroviruses distinguish cis-elements 5'-LTR, 3'-LTR, coat sequence ψ, + and - primer binding site on the spindle of gag (capsid proteins), pol (reverse transcriptase) and env (coat proteins). Transelements can be replaced by foreign genes on the viral genome; however, in this case, the virus relies on so-called helper viruses, which may be either complete, infectious viruses or non-infectious viruses, eg viruses without envelope sequences. Cells containing such non-infectious helper viruses and / or helper genes of one or also of different helper viruses transiently, episomally, or integrated in the genome are referred to as enveloping cell lines. The enveloping cell lines therefore do not release any infectious, replicative virus particles. Where helper genes originate from different helper viruses, the resulting vectors are referred to as chimeric retroviral vectors; this procedure is referred to as pseudotyping. Such chimeric enveloping systems are used in a conventional manner, e.g. GP + env Am12 (Markowitz DG et al. In Trans. Assoc. Am. Physicians 101 (1988) 212-218) carries the gag-pol genes of the ecotropic MoMuLV and the amphotropic amphovirus env-gene. 4070. Env-genes of multiple mixed viruses (even with genetically altered env-genes) may even be present in enveloping cell lines.

The enveloping cell lines may contain incomplete viral helper genomes also fragmented, then the enveloping cell lines with the cleaved viral genome are referred to. They represent a variant with increased certainty against unwanted release of infectious, replicable viruses during the recombination process.

However, if they are transfected or transduced with a retroviral vector which can carry foreign genes in addition to the cis-elements, these viral genomes will be packaged and released as infectious but replicable retroviral vectors. In this case, the cell line of the retrovirus producers is referred to.

All retrovirus enveloped cell lines prepared so far are based on adherently growing, i.e., adhering, surfaces of growing cells:

Markowitz, D.G. et al. in Trans. Assoc. Am. Physicians 101 (1988) 212-218 described GP + E86 and GP + envAml2 enveloping cell lines based on adherent NIH3T3 cells. Miller, A.D. et al. described in Mol. and Cell. Biol. 6 (1986) 2895-2902 a PA317 envelope cell line based on adherent NIH3T3 cells. Miller, A.D. et al., J. Virol. 65 (1991) 2220-2224 described the GALV line of PG13 coat cells based on NIH3T3 cells. Danos, 0 et al. describing • · ···· ····

in PNAS USA 85 (1988) 6460-6464 cell lines in vJ / Cre and vyCrip, based on NIH3T3 cells.

Rigg, R.J. et. al. describes in Virology 218 (1996) 290295 the Propack-A enveloping cell lines which are based on a human cell line. Cosset, F.-L. et al. described in J. Virol. 69 (1995) 7430-7436 FLY A13 and FLY RD18 coat cell lines, which are based on the human fibrosarcoma line (HT1080). WO 92/05266 discloses enveloping cell lines based on D17 and other adherent canine cell lines.

Also, most generally described cell lines grow adherently. Adherent cell lines have the advantage of being easy to work with. In adherent cells it is relatively easy to clone, eventually work with viruses, especially retroviruses, ie clonal selection of cells after transfection eventually transduction is possible directly, individual colonies can be easily identified, counted and pinned, dead cells are easily separated and therefore cannot when replacing the media, it just needs to simply pipette the balance and add new media. Sowing and cell storage are generally not critical, since adherent cells form colonies of cells that stimulate each other as in a tissue bundle. Only in transplanting cells, adherent cells require additional trypsin treatment to separate, but this is not usually critical.

Cells growing in suspension require a different spectrum of methods for cloning and transduction experiments. Clonal selection thus requires working with immobilizing additives such as soft agar, a dilution series of cells known as Limited Dilution, until theoretically only one cell per vessel, or the use of a surface marker or a fluorescent marker (such as GFP) that allow direct selection of recombinant cells using a FAC sorter. Living and dead cells can only be distinguished by staining. Replacing the media always requires a centrifugation step.

Cell lines growing in suspension are not yet known as retrovirus-enveloping cell lines. To date, no experiments have been prepared in which established, adherently growing cell lines in suspension are prepared.

Surprisingly, it appears that the habit of growing in suspension is entirely possible for adherent enveloping cell lines and that the resulting enveloping cell lines are capable of producing high titer viruses. Furthermore, it has also been shown that it is also possible to prepare, in suspension, growing retroviral cell lines by co-transfection of retroviral plasmids with coat genes from classical suspension cell lines, such as the human erytholeukemia cell line K562, rat hepatoma N1-Sl line and rat lymphoma line C58. retroviral vector plasmids and that is, these cells release retroviral vector particles that are capable of transducing other cells.

The preparation of enveloping cell suspension lines is technically analogous to the procedures described many times for adherent cells by introducing helper sequences (as a whole or preferably cleaved) that are not themselves capable of producing an infectious virus, except for the aforementioned difference where suspension cells require other cloning techniques and cultivation.

When a coat cell suspension line is prepared, transduction with a compatible retroviral vector (eg, an amphotropic coat cell line with a vector derived from an ecotropic coat cell line), in particular a transfection with a retroviral vector plasmid, can be converted to a producer suspension cell line, which releases infectious but replication incompetent retroviral vectors.

So far, no mammalian cell lines growing in suspension as retrovirus enveloping cells are known. To date, there are no known methods by which well established, adherently growing enveloping cell lines could be prepared.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide in suspension a growing cell line for retroviral vectors, as well as a process for their preparation and use.

SUMMARY OF THE INVENTION

It is an object of the invention to suspend a fully growing retroviral cell line which is transduced in particular by a retroviral vector.

A further object of the invention is a method for preparing retroviral vectors, characterized in that it is cultured in suspension of a growing cell line that is transduced with said vector, the cells are separated from the cell balance, and the vector is obtained from the balance.

Coating cell line means a mammalian cell line that contains the gag, pol and env genes (helper genes) required for the production of viral particles but does not contain functional active envelope sequences (ψ). Such a cell line cannot itself form any viral particles with genomic RNA.

By functional active envelope sequence is meant a region of the retroviral genome that functionally causes packaging of the RNA genome. Their function can be deactivated by complete or •

• · » - 6 - / « • · • · · · • · · ♦ ··. • 1 · 1 • · · * • · · · · • · · * • · · · · • · · · • · · · partial discarding or mutations. On basis invention are prepared with benefit in suspension completely growing cellular line enveloping retrovirus (i for days, for example it is not

no insoluble matrix (vessel wall or microcarrier to which cells adhere) to grow from adherently growing retrovirus-enveloping cell lines by adapting such an adherent enveloping cell line over a period of at least three months step by step to grow in suspension. The steps include continuous reduction of FKS content in the medium to absence of serum, re-treatment of cells with trypsin and / or DNAse, using medium for suspension cell lines (e.g. DMF024A), especially tissue culture bottles for suspension and hybridoma cells (Sarstedt), continuous rocking shake flasks as well as cell densities between 5 χ 10 ⁴ and 1 χ 10 ⁶ cells / ml (this results in significantly more frequent cell cleavage than comparable adherent cultures).

According to the invention, a cell line completely grown in suspension, which is transfected with or transduced with a retroviral vector, is preferably prepared by suspending a cell line that is transfected with a retroviral vector plasmid or transduced with a retroviral vector, the transfected or transduced cells selected with by limiting dilution and / or adding immobilizing additives and the thus selected suspension cells are isolated. Preferably, the suspension cell line is a retrovirus enveloping cell line. For preparing in suspension a growing retrovirus-enveloping cell line is preferably an adherently growing retrovirus-enveloping cell line that is transfected with a retroviral vector plasmid and nucleic acids that encode

helper sequences, or transduced with a retroviral vector and cultured in serum-containing medium, transferred to a cell line growing in suspension by continuously reducing the serum content of the medium until complete serum release, retreating the cells with trypsin and DNAse to separate the cells from the carrier. the cell density in the suspension is maintained between 5 χ 10 ⁴ and 1 χ 10 ⁶ cells / ml. Preferably, the process is preferably carried out over a longer period of time, in particular at least three months. In another preferred embodiment, the transfected or transduced cells are selected by a co-transfected or co-reduced surface marker by limited dilution and / or by the addition of immobilizing additives.

Surprisingly, it has been shown that, despite a massive long-term manipulation lasting several months, the thus treated suspension of the completely growing enveloping cell line is still able to produce retroviruses after being transfected with the retroviral vector plasmid and transduced with the retroviral vector, respectively.

Just as surprisingly, these fully growing cell lines in suspension can be cloned by transfection with retroviral vector plasmids or transduction with retroviral vectors by separation steps such as limited dilution, in which the cells are so diluted that only single cells are practically present in the suspension per container, or by using immobilizing additives such as coating agar or methylcellulose (Metho Cult H4100 / Stem Cell Technologies) or sorting cells against cell surface marker, and then emerging in suspension of fully growing retrovirus producer cell lines capable of producing retroviral vectors on the same scale as adherent retrovirus producer cell lines.

retrovirus producers

The retrovirus producer cell lines thus prepared allow substantially simplified large-scale cultivation in stirred fermenters or the High-Density Dialysis cultivation method as compared to the adherent retrovirus producer cell lines.

Furthermore, methods of obtaining vector viruses are distinguished. While in adherent retrovirus producer cell lines, retrovirus-containing culture residues must only be removed, while a sterile filtration step, although not urgently needed, is nevertheless performed for safety reasons, much smaller, lighter vector retrovirus particles must be separated from the fully growing cell line suspension. producing cell lines from larger, heavier by filtration and centrifugation steps or at least 2 filtration steps.

Retroviral vector plasmids or retroviral vectors are those retroviral vectors that contain a therapeutic gene. These vectors contain no coat sequences such as gag, pol, env to prevent the release of replication-capable retroviral vectors in the production of suspension producer cell lines. Typically, a therapeutically relevant gene is inserted between the 5'-LTR and the 3'-LTR, with the selection genes suitably comprising a neomycin or hygromycin resistance gene.

Replication-incompatible (replication-defective) vector means a vector that does not contain any retroviral gene functions (gag, pol, env) and therefore cannot form any single viral particles. Usually, however, the vector contains a packaging function (ψ). To create infectious • retroviral particles! a so-called " packaging cell line " which contains the gag, env and pol gene functions stably (as an episome or integrated into the genome) is transduced with a replication-defective DNA vector. RNA transcripts are produced, which are packaged into viral particles based on gag functions and enfunctions. These viral particles are replication deficient because the RNA genome contained therein carries no retroviral gene functions.

DETAILED DESCRIPTION OF THE INVENTION

The following examples and publications further illustrate the invention, the scope of protection of which is set forth in the claims. The procedures described are to be understood as examples which, after modification, describe the subject matter of the invention.

Example 1:

Adaptation of adherent cell lines enveloping retroviruses to grow in suspension

The adherent cell line of FLY A13 (amphotropic env from MoMuLV) and FLY RD18 (env from cat endogenous RD114) (Cosset, F.-L. et al. J. Virol 69 (1995) 74307436), both of which are both based on the HT1080 human fibrosarcoma line, the following steps were adapted to grow in suspension:

(a) Adaptation of adherent enveloping cell lines to suspension growth:

I. continuous and slow FKS-reduction in medium; for about 5 months;

10% FKS-> 5% -> 2.5% -> 2% -> 1% -> 0.5% -> serum free

II. use of medium for suspension cell lines

III. use of culture flasks for suspension cell lines · ··:

- ίο -. ·. . _: ·:: ·: .....

.: ...... · Sar **

IV. continuous, light shaking culture flasks on a shaker

V. storage requires dissolution of the resulting cell clumps in trypsin and treatment with DNAse and more frequent cleavage to remain in suspension.

Cell density is always a critical factor (5.10 ⁴ - 1.10 ⁶ cells / ml), ie it must be neither too high nor too thin.

b) Determination of retrovirus titer in transient cultures

In suspension, the growing enveloping cell lines were compared to the corresponding adherently growing enveloping cell lines with respect to the production behavior of the retroviral vectors.

Therefore, all cell lines were transfected with a retroviral vector plasmid and the titer of retroviruses in transient cultures was determined.

Material:

* Retroviral vector pLANSN (6853bp) [-5'LTR MoMuLV-ψ -ALNGFR-SV40-P / E-neoR-3'LTR MoMuLV-pBR322-plasmid fraction] Transfection reagents; DOSPER; 20 μg DOSPER / batch each time (manufacturer: Boehringer Mannheim GmbH, Germany) * DNA concentration in plasmid: 5 gg DNA / batch / plasmid

Methods:

day 1: cells seeded for transfection:

Adherent cells: in 60 mm Petri dishes each 6x10 ⁵ cells / suspension cell dish: in suspension cell culture flasks; (T25, Sarstedt), plated, up to 3x10 ⁵ cells / ml each; total together 2 ml.

Incubate overnight at 37 ° C, 5% CO 2, suspension cells were gently shaken.

Day 2: change media (for suspension cells by centrifugation and resuspension), prepare DNA / DOSPER-Mix and drip onto cells (100 μΐ DNA / DOSPER-Mix).

Incubate for 5-6 h, 37 ° C, 5% CO ₂ . Finally, the medium was added.

Day 3: 24 h after transfection, the residue was harvested, centrifuged (only on suspension cell lines!), Filtered (0.45 µm filter) and titrated to NIH3T3 cells (G418 selection).

Result of titration about 10-14 days later.

Titr:

adherent FLY A13 cell lines: 8x10 ⁵ cfu / ml

2x10 ⁵ cfu / ml

FLY RDI8: cell lines suspended:

FLY A13 2x10 ³ cfu / ml

FLY RD18 1x10 ² cfu / ml

Result:

Suspended enveloped cell lines are principally capable of producing recombinant retroviruses.

The differences in titer can be derived from different transfection conditions and titer determination, for example, different transfection efficiencies in suspension cells and adherent cells, and different stability or infectivity of retroviruses in FKS-free and FKS-containing medium.

Example 2:

Retrovirus production after transient triple transfection of both helper plasmids and retroviral vector plasmid in suspension cell lines

One adherently and several suspension-growing cell lines were compared in transient triple transfection by the ability to form infectious retroviral vectors.

Cell lines used:

adherent cell line (control) NIH3T3 (mouse fibrosarcoma) suspension cell line: K562 (human erythroleukemia cell line)

Nl-Sl (rat hepatoma)

C58 (rat lymphoma)

Material:

(a) Auxiliary plasmids:

• gag-pol plasmid expression pGAPOGPT (10202 bp) [-CMV-P / E-gag-pol-polyA-SV40-P / E-gpt-polyA-pUC19 plasmid fraction]

ENV-plasmid expression of pENVCHT (7933 bp) [-TK-P-hygR-polyA-CMV-P / E-env (amphotrope) -I polyA-

pUC20-plasmid fraction]

(b) Retroviral vector plasmid:

• Retroviral vector pLANSN (6853 bp) [-5'LTR MoMuLV -] / - ALNFGR-SV40-P / E-neoR-3'LTR MoMuLVpBR322-plasmid fraction] Transfection reagent: DOSPER (BM); always 20 pg

DOSPER / handpiece • Plasmid DNA concentration: 5 µg DNA / handpiece / plasmid

Methods:

Day 1: Cells seeded for transfection:

adherent cells (NIH3T3): in 60 mm Petri dishes each 6x10 ⁵ cells / suspension cell dish: in T25 culture flasks (for suspension cells, upside down), 3x10 ⁵ cells / ml each; total 2 ml.

Incubate overnight at 37 ° C, 5% O ₂ / suspension cells were gently shaken.

Day 2: Change media (for suspension cells by centrifugation and resuspension), prepare DNA / DOSPER mix and drip onto cells (100 μΐ DNA / DOSPER mix each time).

Incubate 5-6h, 37 ° C, 5% CO ₂ . Finally, the medium was added.

Day 3: 24 h after triple transfection, harvest the residue, centrifuge (only on suspension cell lines!), Filter (0.45 μη filter) and titrate to NIH3T3 cells (selection G418).

Result of titration about 10-14 days later.

• ·

Result of titration on NIH3T3 (number of G418-resistant colonies):

NIH3T3 (adherent control cells): 3 colonies per 1.4 ml virus balance (titer: 2 cfu / ml)

K562: 6 colonies per 0.8 ml virus balance (titer:

7.5 cfu / ml)

N1-Sl: 6 colonies per 2 ml virus balance (titer: 3 cfu / ml)

C58: 1 colony per 2 ml virus balance (titer: 0.5 cfu / ml)

List of references

Cosset, F.-L. et al., J. Virol. 69 (1995) 7430-7436

Danos, O. et al., PNAS USA 85 (1988) 6460-6464

Markowitz, D.G. et al., Trans. Assoc. Am. Physicians 101 (1988) 212-218

Miller, A.D. et al., J. Virol. 65 (1991) 2220-2224

Miller, A.D. et al., Mol. and Cell. Biol. 6 (1986) 2895-2902. Rigg, R.J. et al., Virology 218 (1996) 290-295

Claims (3)

A method for preparing a suspension of a completely growing enveloping cell line, characterized in that the adherently growing enveloping cell line, which is transfected with nucleic acids encoding helper sequences and a retroviral vector plasmid or is transduced with a retroviral vector, and cultured in serum-containing medium is transferred to a cell line growing in suspension by continuously reducing the serum content of the medium until serum is released, retreating the cells with trypsin and DNAse to separate the cells from the carrier, maintaining the cell density in the suspension at 5 χ 10 ⁴ 1 χ 10 ⁶ cells / ml. Method according to claim 1, characterized in that the process is carried out for a period of at least three months. Method according to claim 1 or 2, characterized in that the transfected or transduced cells are selected by a cotransfected or cotransduced surface marker by means of limited dilution or addition of immobilizing additives.