U.S. patent application number 12/522646 was filed with the patent office on 2010-01-07 for production of lentiviral vectors.
Invention is credited to Kari J. Airenne, Hanna P. Lesch, Seppo Yla-Herttuala.
Application Number | 20100003746 12/522646 |
Document ID | / |
Family ID | 37899177 |
Filed Date | 2010-01-07 |
United States Patent
Application |
20100003746 |
Kind Code |
A1 |
Lesch; Hanna P. ; et
al. |
January 7, 2010 |
Production of Lentiviral Vectors
Abstract
The present invention is a method of generating a lentivirus
vector, comprising cloning each of a leotivimus transfer construct,
gag, pol, an envelope protein and rev respectively into the same or
different baculoviruses, and transducing a producer cell with the
or each baculovirus.
Inventors: |
Lesch; Hanna P.; (Kuopio,
FI) ; Airenne; Kari J.; (Kuopio, FI) ;
Yla-Herttuala; Seppo; (Kuopio, FI) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Family ID: |
37899177 |
Appl. No.: |
12/522646 |
Filed: |
February 11, 2008 |
PCT Filed: |
February 11, 2008 |
PCT NO: |
PCT/GB2008/000464 |
371 Date: |
July 9, 2009 |
Current U.S.
Class: |
435/320.1 |
Current CPC
Class: |
A61K 48/0091 20130101;
C12N 2710/14043 20130101; C12N 2740/16022 20130101; C12N 2740/16045
20130101; C12N 2740/16043 20130101; C12N 15/86 20130101; C12N
2740/16051 20130101; C12N 2740/15051 20130101; C12N 2810/6081
20130101; A61K 48/00 20130101; C07K 14/005 20130101; C12N
2710/14051 20130101; C12N 2740/15043 20130101; C12N 7/00 20130101;
C12N 2710/14021 20130101 |
Class at
Publication: |
435/320.1 |
International
Class: |
C12N 15/79 20060101
C12N015/79 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 12, 2007 |
GB |
0702695.8 |
Claims
1. A method of generating a lentivirus vector, comprising cloning
each of a lentivirus transfer construct, gag, pot an envelope
protein and rev respectively into the same or different
baculoviruses, and transducing a producer cell with the or each
baculovirus.
2. The method according to claim 1, wherein the envelope protein is
VSV-G.
3. The method according to claim 2, wherein the lentivirus transfer
construct, gag, pol, VSV-G and rev are respectively cloned into
BAC-transfer, BAC-gag-pol, BAC-VSVg and BAC-rev.
4. The method according to claim 1, wherein the lentivirus transfer
construct, gag, pol, the envelope protein and rev are each cloned
into the same baculovirus.
5. The method, according to claim 1, wherein the producer cell is
293T, HepG2, CHO, BHK, Sf9, Sf21, 293,BTI-Tn 5 B 1-4, COS, NIH/3T3,
Vero, NSO or PerC6 cells.
6. The method according to claim 1, wherein the lentivirus transfer
construct is a third-generation lentivirus transfer construct.
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods for the production of
lentiviral vectors.
BACKGROUND OF THE INVENTION
[0002] Lentiviruses, such as Human Immunodeficiency Virus I, are
promising tools for gene therapy due to their ability to transduce
and integrate into the genome of both dividing and non-dividing
cells. However, the production of replication-defective lentiviral
vectors for large-scale clinical use is challenging. Lentiviral
vectors are normally produced by cotransfecting 293T human
embryonic kidney cells with several different plasmid constructs.
The first clinical lentiviral vector production was based on a
two-plasmid system (Lu et al., 2004). To further improve the safety
of the system, lentivirus genome can be separated into four
plasmids. The plasmids are a self-inactivating transfer vector; a
packaging plasmid containing gag-pol; a rev plasmid; and an
envelope glycoprotein plasmid which usually encodes vesicular
stomatitis virus glycoprotein G (VSV-G). To upscale lentivirus
production, the growth of adherent cells has been changed to cell
factories. Lentiviral vectors have also been transiently produced
in suspension cultures using 3-L bioreactors in serum-free
conditions.
[0003] As transient transfection systems for virus production can
be problematic and time-consuming, attempts have been made to
develop stable large-scale production systems. However, the
toxicity of lentiviral protease and the fusogenic envelope protein,
VSV-G, has prohibited constitutive vector production. One
production method uses inducible packaging cell lines, controlled
either by tetracycline- or ecdysone-inducible promoter systems.
Other production methods involve replacing the toxic VSV-G protein
with less toxic glycoproteins.
[0004] Lentiviral vectors can also be pseudotyped by various viral
surface proteins. The most commonly used is the envelope
glycoprotein G of the vesicular stomatitis virus (VSV-G). In
addition, various different envelope glycoproteins such as those
from gamma-retroviruses (e.g. feline endogenous retrovirus RD114
env, modified gibbon ape leukaemia virus GaIV, moloney murine
leukaemia virus MLV), alphaviruses, lyssaviruses or baculoviruses
had also been shown to pseudotype lentiviral vectors. Pseudotyping
broadens the transduction range of lentiviruses, and long-term
transgene expression has been obtained in many different cells and
tissues (Delenda, 2004). Pseudotyping also strengthens fragile
lentiviruses and enables concentration to high titers by
ultracentrifugation.
[0005] Baculoviruses possess several advantages for gene delivery
applications. They have a large insert capacity (>100 kb) and
are capable of transducing most mammalian cell lines, even in
large-scale suspension cell cultures in serum-free conditions
(Scott et al., 2007). In addition, baculoviruses are easy to
produce in large-scale and high titers, and they present few safety
problems as they are not able to replicate in mammalian cells.
Cytotoxicity is very rarely detected, even with high Multiplicity
of Infection (MOI).
[0006] Baculoviruses have been used for a large-scale protein
production in insect cells and for the production of viral-like
particles (VLP), such as hepatitis VLP. Intact viruses have also
been produced using hybrid baculoviruses. Baculovirus-mediated
production of recombinant influenza viruses, adenoviruses
(Cheshenko et al, 2001) and AAV (Auang et al, 2007) in mammalian
cells has also been described.
SUMMARY OF THE INVENTION
[0007] According to the present invention, a method of generating a
lentivirus vector, comprises cloning each of a lentivirus transfer
construct, gag, pol, an envelope protein and rev respectively into
the same or different baculoviruses, and transducing a producer
cell with the or each baculovirus.
DESCRIPTION OF THE DRAWINGS
[0008] The following drawings illustrate embodiments of the
invention.
[0009] FIG. 1 is a schematic diagram and shows cloned baculovirus
donor plasmids, BAC-transfer, BAC-gag-pol, BAC-VSVg and
BAC-rev.
[0010] FIG. 2 is a schematic diagram of baculovirus-mediated
lentivirus production.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0011] To perform the method of the invention, the baculovirus(es)
must comprise a lentivirus transfer construct, gag, pol, a suitable
envelope protein and rev.
[0012] The term "lentivirus transfer construct" will be known to
those skilled in the art. A lentivirus transfer construct is a
source of lentiviral RNA genome/lentiviral vector RNA and transgene
cassette. One example of a lentivirus transfer construct is
LV1-GFP. Methods for producing lentivirus transfer constructs will
also be known to those skilled in the art.
[0013] The term "envelope protein" will be known to those skilled
in the art. An envelope protein is a protein that protects the
nucleic acid of a virus. An example of envelope protein suitable
for use, in the invention is VSV-G.
[0014] The term "producer cell" will be known to those skilled in
the art. Examples of producer cells suitable for use in the
invention are 293T, HepG2, CHO, BHK, Sf9, Sf21, 293, BTI-Tn 5 B
1-4, COS, NIH/3T3, Vero, NSO or PerC6 cells. The produced cells may
then be cultured as adherent or in suspension.
[0015] Preferably, all the elements needed for the production of
functional lentiviruses according to the invention are cloned into
three different baculoviruses. More preferably, they are cloned
into two different baculoviruses, and most preferably, they are
cloned into one single baculovirus. This may be achieved by
combining the features of BAC-transfer, BAC-gag-pol, BAC-VSVg and
BAC-rev into a single baculovirus.
[0016] Four recombinant baculoviruses BAC-transfer, BAC-gag-pol,
BAC-VSVg and BAC-rev, derived from Autographa califomica
multicapsid nucleopolyhedrovirus (AcMNPV) have been constructed.
They encode all the elements needed for lentivirus vector
generation in mammalian cells. By transducting 293T cells with
these baculoviruses, a functional lentivirus has been produced.
[0017] Baculovirus technology is an attractive option for scalable
virus production due to ease of production, concentration of
baculovirses, efficient transduction of suspension mammalian cells
in serum-free conditions and safety. The lentiviral vector may also
be produced with the baculovirus encoding or displaying viral
protein rev, tat, net, vit or vpu, by, but are not limited to,
fusing the viral protein to a baculovirus protein. The baculovirus
protein may be the major envelope protein gp64 or the capsid
proteins, vp39 and p24.
[0018] The resulting lentiviruses may be pseutotyped with
heterologous proteins or other ligands, such as VSV-g, gp64,
avidin, streptavidin or biotin.
[0019] The following Example illustrates the invention.
Materials and Methods
Cloning of Plasmids for the Production of Baculoviruses
[0020] All the necessary elements for the production of 3.sup.rd
generation lentiviral vectors in mammalian cells were subcloned
into baculovirus donor vector pFastBac1 to construct four
recombinant baculoviruses, BAC-transfer, BAC-gag-pol, BAC-VSVg and
BAC-rev, derived from Autographa californica multipled
nucleopolyhedrovirus (AcMNPV) (FIG. 1). First, a polylinker
containing multiple cloning sites
(PmlI/NheI/PstI/SalI/AflII/PacI/SpeI/MluI/Pmel/EcoRI/Apal/Swal/AscI)
was cloned into the unique AvrII site of pFastBac1. The sequence of
the polylinker was 5'
CACGTGGCTAGCCTGCAGGTCGACCTTAAGTTAATTAAACTAGTACGCGTGT
TTAAACGAATTCGGGCCCATTTAAATGGCGCGCC-3' (SEQ ID No: 1). The donor
vector also contained the red fluorescent protein marker gene
(DsRed) under the control of a polyhedrin promoter for convenient
baculovirus titer determination.
[0021] To generate a third-generation self-inactivating lentivirus
transfer construct, (LV1-GFP) .DELTA.NGFP from plasmid
LV-hPGK-.DELTA.NGFP-WPRE-SIN (Makinen, 2006) was replaced by GFP.
In this construct, the GFP marker gene is driven by the
phosphoglycerate kinase (PGK) promoter. The pBAC-transfer vector
was constructed by subcloning the relevant sequence from LV1-GFP
into the pFastBac1 donor vector polylinker in two stages. To clone
the first part of the sequence, LV1-GFP was digested with BsrBI and
AscI and subcloned into SwaI/AscI-site of the donor vector
polylinker. The second part of the sequence was cloned by digesting
LV1-GFP with AscI and AvrII and inserting the fragment into the
AscI and AvrII sites of the modified pFastBac1-plasmid.
[0022] The packaging construct (pBAC-gag-pol) expressing gag and
pol driven by CMV promoter was derived from the plasmid pMDLg/pRRE
(Follenzi and Naldini, 2002) by ApaLI digestion and subcloned into
the SmiI-site of the donor vector. Prior to ligation ApaLI ends
were blunted with T4 DNA Polymerase (Finnzymes, Helsinki,
Finland).
[0023] The VSV-G envelope construct from the plasmid pCMV-VSVG was
subcloned into the pFastBac1 vector in two stages. First, pCMV-VSVG
was digested with NotI and blunted using T4 DNA Polymerase prior to
digestion with EcoRI. This fragment was subcloned to the
SmiI/EcoRI-site of the polylinker. The second part of the sequence
was digested from PCMV-VSVG with EcoRI and subcloned into the
polylinker EcoRI site.
[0024] Rev cDNA was obtained by polymerase chain reaction (PCR)
from the plasmid pRSV-REV (Dull et al, 1998) using forward and
reverse primers. The forward primer is 5'
CGAAGGAATTCGTCGCCACCATGGCAGGAAGAAGCGGA-3' (SEQ ID No: 2). The
sequence for nucleotides 1-18 of the rev gene is in bold, the Kozak
concensus sequence is in italic, and the EcoRI site underlined. The
reverse primer is 5' AGCTAGCTAGCGTATTCTCCTGACTCCAATATTGT-3' (SEQ ID
No: 3). The sequence for nucleotides 349-325 of the rev gene is in
bold and the NheI site is underlined. The amplified PCR product was
digested with EcoRI and NheI, purifed using a Wizard Clean up kit
(Promega, Madison, Wis., USA), and subcloned into the
EcoRI/NheI-site of the pFastBac1 polylinker to form pBAC-rev. Rev
cDNA was under the control of the CMV promoter which was previously
subcloned as a NruI/EcoRI fragment from the pcDNA3 vector
(Invitrogen) into the SwaI/EcoRI-site of the pFastBac1
polylinker.
Production of Lentiviruses
[0025] 293T cells were plated 24 h before transduction. Cells were
cultured in Dulbecco's Modified Eagles Medium (DMEM) or RPMI 1640
both supplemented with 10% fetal bovine serum (FBS). Transduction
was performed with varying multiplicity of infection (MOI) between
50 and 1000 pfu per cell in either serum-free or serum supplemented
DMEM or RPMI. After 4 h incubation in serum-free medium or 18 h in
serum supplemented DMEM or RPMI at 37.degree. C. the cells were
washed and medium was changed. The cell supernatant containing
lentiviruses was collected 48 h post transduction and centrifuged
at 1500 rpm for 10 min at room temperature.
[0026] As controls, batches were made where each of the three
baculoviruses were missing (BAC-gag-pol, BAC-Rev or BAC-VSVg).
Lentiviruses were also prepared by conventional four plasmid
transient transfection method in 293T cells (Follenzi and Naldini,
2002). To improve the attachment of the cells to the bottom of the
plates, the plates were coated with poly-L-lysine, according to the
manufacturer's instructions.
Titering of Lentiviruses
[0027] Transforming units of lentiviruses (TU/ml) were determined
by analyzing the number of virus particles able to transduce HeLa
cells. On day one Hela cells were seeded in 6-well plates at
1.times.10.sup.5 or in 96-well plates at 5.times.10.sup.3 cells per
well. The lentivirus transduction was carried out on day two with
serial dilution. On day five the cells were visualized with
fluorescent microscopy and analyzed by flow cytometry to reveal the
percentage of cells which were transduced by GFP expressing
lentiviruses. Titers were calculated as described in Follenzi and
Naldini, 2002.
Long Term Expression of Transgene
[0028] HeLa cells (5.times.10.sup.3) were seeded on the 96 well
plates, transduced next day with baculovirus-produced lentiviruses
and cells were cultured up to 6 weeks. GFP expression was monitored
weekly by flow cytometer. As an additional control, Hela cells were
also transduced with baculovirus BAC-transfer expressing GFP and
the expression was monitored in a similar way.
Determination of p24 Concentration
[0029] The amount of lentiviral capsid protein p24 (pg/mi) was
determined by p24 ELISA kit (NEN.TM. Life Science Products HIV-1
p24 ELISA). Testing of the replication competent lentiviruses (RCL)
was done by p24 ELISA determination from the cell culture
supernatants. HeLa cells were transduced with lentivirus and the
transduction efficiency was monitored by flow cytometer. Cells were
cultured for four weeks, supernatants were collected and the
concentration of p24 in the supernatants was measured repeatedly as
a marker of RCL. This was further confirmed by transducing naive
HeLa cells with collected supernatants and GFP expression was
monitored by fluorescent microscopy and flow cytometer.
Statistical Analyses
[0030] Statistical analyses were performed by GraphPadPrism 4
(GraphPad Software Inc., San Diego, Calif., USA).
Results
Construction of Baculoviruses
[0031] The key 3.sup.rd generation elements were cloned into four
baculoviruses. The baculovirus plasmid constructs were verified by
restriction analysis and four baculoviruses were generated in
Insect cells. To monitor consistency of the baculovirus production,
immunoblot analysis was conducted on each batch, with anti-gp64
against major envelope protein of baculovirus. End point titer
determination (IU/ml) for concentrated baculoviruses was done in
insect cells. High titers (>1010 IU/ml) were measured for all
produced viruses and used to control MOI in lentivirus
production.
Production of Lentiviruses
[0032] Lentiviruses were produced by transduction of 293T cells
with four baculoviruses. Transduction was performed using different
mediums and incubation times. The transduction efficacy was
monitored 20 h after the transduction by fluorescent microscopy.
Lentivirus containing supernatants were collected 48 h after the
transduction and titers were determined in HeLa cells as
transducing units (TU/ml).
[0033] Four baculoviruses at MOI 500 each yielded lentivirus titers
with an average of 6.0.times.10.sup.5 TU/ml when transduction was
performed 4 h in serum-free conditions. Baculovirus concentration
at MOI 750 produced higher lentivirus titers with an average of
1.2.times.10.sup.6 TU/ml. A decrease in titer was detected when
higher baculovirus concentrations (four baculoviruses at MOI 1000
each) were used. Four baculoviruses at MOI 250 yielded the highest
titers with an average of 2.5.times.10.sup.6 TU/ml when baculovirus
transduction was performed over night. When RPMI medium was used in
transduction, the highest titers were an average of
5.9.times.10.sup.5 TU/ml already at MOI 50. The titers are
comparable to those produced with conventional four plasmid
transduction method (Follenzi and Naldini, 2002).
[0034] The different ratio of plasmids can influence titers in
plasmid transfection. When the same ratio of baculoviruses as is
commonly used with plasmids were used, the lentivirus titers were
0.64 fold lower than expected. BAC-transfer virus was also doubled,
to see if higher lentivirus titers could be obtained. However,
there was no significant difference in the titers compared to the
production with similar doses of baculoviruses. Titers obtained
with the doubled amount of the BAC-transfer were at on an average
of 1.4.times.10.sup.6 TU/ml.
[0035] As negative controls, lentivirus production was performed,
omitting one of the baculoviruses (BAC-gag-pol, BAC-Rev or
BAC-VSVg) at a time. Collected mediums were used to transduce HeLa
cells and the number of GFP positive cells (%) was analyzed four
days after the transduction by flow cytometer. No GFP positive
cells could be determined in these experiments.
[0036] A frequently used titration method alongside with a
biological titer (TU/ml) of lentiviruses measures p24 concentration
(pg/ml) by ELISA. The p24 concentrations in the medium were
191.+-.105 ng/ml which corresponds the values for the
representative virus preparations. However, p24 concentration does
not separate biologically active particles. To compare infectious
particles and p24 ratio, both of these parameters were measured
from several preparations produced with different amounts or ratios
of baculoviruses. The results showed good TU/p24 ratio.
Characterization of Transgene Expression
[0037] Residual baculoviruses in the collected lentivirus medium
was evaluated by end point tittering and the titer was 0.1-0.5%
from the dose used for 293T cell transduction. To confirm that
transgene expression was originated from the produced lentivirus,
and not from residual baculovirus, 293T cells were transduced with
the BAC-transfer baculovirus only. HeLa cells were then transduced
with the medium collected similar way than in lentivirus
preparation and GFP positive cells were analysed with flow
cytometer four days after transduction. No GFP positive cells could
be detected.
[0038] Baculovirus vectors do not replicate in vertebrate cells and
they cannot integrate into the host genome. Gene expression from
these vectors is transient and usually lost in two weeks. However,
transgene expression from an integrated lentivirus is relatively
stable assuming no silencing of the transgene expression occurs.
Baculovirus-produced lentiviruses transduction lead to efficient
GFP expression which could still be observed after 43 days post
transduction (FIG. 5A). Expression was also detected by fluorescent
microscopy in HeLa cells at day three Baculovirus-mediated GFP
expression at MOI 100 and 1000 (18.7+1.9% and 11.5.+-.0.4% at day
three, respectively) was lost 17 days after post transduction.
Replication-Competent Lentiviruses
[0039] Replication-competent lentiviruses (RCL) were tested by p24
ELISA assay. HeLa cells were transduced with lentivirus containing
mediums. Transduction efficiencies were verified by flow cytometer.
Cells were cultured for four weeks and concentration of p24 in the
supernatant was repeatedly measured. An increasing concentration of
p24 would indicate an ongoing viral replication, but no such
increase was detected. Mediums collected from transduced HeLa cells
after 2.5 weeks were further used to transduce naive HeLa cells but
no GFP expression was detected neither with fluorescent microscopy
nor flow cytometer.
[0040] In summary, successful generation of functional lentiviruses
using hybrid baculoviruses has been demonstrated. Lentivirus titers
produced by baculoviruses were comparable to those produced using
the conventional four plasmid transfection method. Good lentivirus
titers were achieved when optimal dose of baculoviruses and
extended transduction time was used. A decrease in lentivirus
titers and cell death was observed when high doses of baculoviruses
were used. This may be due to the VSV-G toxicity to production
cells. No problems were observed when the VSV-G expressing
baculovirus was omitted, keeping the total number of baculovirus
particles constant. By replacing the DMEM medium with RPMI1640, the
lowest baculovirus dose (MOI 50) resulted in the best lentivirus
titers. This is in line with the fact that the transduction medium
affects baculovirus-mediated gene expression in vertebrate cells.
To confirm the functionality of the generated lentiviruses, HeLa
cells were transduced and sustained GFP expression was observed for
6 weeks. However, with the control baculovirus the GFP expression
was lost in 17 days. If lentivirus generation was performed by
omitting either BAC-gag-pol, BAC-Rev or BAC-VSVg, no lentivirus was
produced.
[0041] Although baculoviruses are safe, contamination of the
lentivirus preparation with baculoviruses is not desirable. The
amount of residual baculoviruses in the lentivirus preparations was
only 0.1-0.5% of the baculovirus dose used in the simple lentivirus
production protocol in which the 293T cells were washed only once
after baculovirus transduction. The residual baculovirus may be
further reduced by simply adding extra washing step(s) or using
adequate down-stream purification schema.
[0042] One of the major concerns associated with the use of
lentiviral vectors is the probability of generating pathogenic
human viruses. To avoid this, the lentivirus genome was separated
into four different production plasmids in order to minimize the
risk of RCL formation by recombination. No RCL was detected fin the
baculovirus-generated lentivirus preparations. p24 levels were not
increased after prolonged cultures and no GFP expression was
detected in 2.5 weeks after transduction.
[0043] The scalability of virus production for clinical studies
remains difficult in adherent cells. Thus, adaptation of lentivirus
production to suspension cell cultures would be advantageous.
Preliminary results in suspension adapted HEK293 cells in
serum-free conditions showed very efficient baculovirus
transduction efficacy (95.1% GFP positive cells).
REFERENCES
[0044] Delenda, 2004. J. Gene Med. 6 Suppl 1: S125-S138. [0045] Lu,
et al. J. Gene Med. 6, 963-973 (2004). [0046] Follenzi, &
Naldini Methods Enzymol. 346, 454-465 (2002). [0047] Ni, et al. J.
Gene Med. 7, 818-834 (2005). [0048] Cheshenko, et al, Gene Ther. 8,
846-854 (2001). [0049] Makinen, et al. J. Gene Med. 8, 433-441
(2006). [0050] Dull, et al. J. Virol. 72, 8463-8471 (1998).
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