Download Report Andre Manrique LV

CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAIS
LABORATÓRIO NACIONAL DE BIOCIÊNCIAS
LABORATÓRIO VETORES VIRAS- LVV
21º PROGRAMA BOLSAS DE VERÃO
OPTIMIZATION OF PARAMETERS FOR RAPID AND ACCURATE TITRATION OF
LENTIVIRAL VECTORS
ORIENTADOR: MARCIO CHAIM BAJGELMAN
ANDREA JHOANNA MANRIQUE RINCÓN
CAMPINAS-SP
2012
“It is not the possession of truth, but the success which attends the seeking after it, that
enriches the seeker and brings happiness to him.”
MAX PLANCK
2
Acknowledgment
I owe my sincere thanks to CNPEM, LNBIO and LVV for the support to this project and
also for the wonderful experience in science that it brings to me.
I want to express my gratitude to my adviser Marcio Chaim Bajgelman for allowing me to
be involved in this research, share with me his knowledge in science and for all his
valuable comments that makes me more conscious about the scientific work that I want to
do.
I thank Anna Carolina Pereira Vieira de Carvalho,MSc for technical assistance and Maria
Eugênia Ribeiro de Camargo for the assistance with flow cytometry.
I want to thank also to Juan José Builes, Regina Cicarelli, Isabel Ortiz, Juan Pablo
Narváez, my family, friends and Professors in University of Antioquia, for their support
during this time.
Finally to my new friends from this program whose backing was really important for me.
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TABLE OF CONTENTS
ACKNOWLEDGMENT
3
ABSTRACT
5
INTRODUCTION
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RETROVIRUS FEATURES AND GENE TRANSFER
LENTIVIRAL VECTORS : IMPROVING GENE TRANSFER EFFICIENCY
EVALUATING TRANSDUCTION EFFICIENCY AND CONCENTRATION OF A VIRAL PREPARATION
NON FUNCTIONAL METHODS
P24 antigen ELISA:
Reverse transcriptase activity
Determination of the genomic RNA concentration
FUNCTIONAL METHODS
Lentiviral vector titration from transgene protein expression
Flow cytometry
Quantitative Polymerase chain reaction
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10
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OBJECTIVES
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SPECIFIC AIMS
12
MATERIAL AND METHODS
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CELL LINES AND CULTURE CONDITIONS
VIRUS PRODUCTION
VIRUS TRANSDUCTION
FLOW CYTOMETRY
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13
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14
RESULTS AND DISCUSSION
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DETERMINATION OF LINEAR RANGE FOR VIRAL DILUTIONS
LEVEL OF REPORTER EXPRESSION IS CRUCIAL FOR TITRATION ACCURACY
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ADDITIONAL EXPERIMENTS
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BIBLIOGRAPHY
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4
Abstract
The use of virus for the introduction of DNA is a technique that has been widely
used to the establishment of permanent cell lines, to produce transgenic animals, to study
RNAi, to analyze gene function, to produce recombinant proteins and for gene therapy.
One of the most commonly used vectors are lentivirus, because of their high
efficiency of integration in the host genome of both dividing and non-dividing cells, their
low immunogenicity, and the fact that they can be extensively modified to produce
recombinant particles. Lentivirus can be transiently produced by co-transfection of
plasmids encoding viral genome and packaging plasmids. After transfection, viral particles
are produced, released in the supernatant and can be purified.
A critical issue regarding virus production is to determine the viability and
concentration of viral preparation. A number of methods to evaluate lentiviral titer have
been described. Titration can be performed quantifying viral antigens as p24 by Elisa, or
using a biological method that allows an accurate detection of functional particles, based
on antibiotic selection, detection of reporter genes by flow cytometry or quantifying
transducing units using qPCR. All titration methods have advantages and limitations that
should be considered.
In this work we studied the optimization of a biological titration method based on
flow cytometry for detection of a fluorescent reporter gene. We used a lentiviral vector
encoding GFP to transduce target cells that were analyzed by flow cytometry, testing
parameters as virus dilution, time of incubation to detect reporter gene and efficiency of
transduction using different cell lines. Our data revealed some aspects applicable to
experimental protocols, to enhance titration accuracy using flow cytometry.
5
Introduction
Retrovirus features and gene transfer
Retroviruses belong to the large Retroviridae family. The virions are roughly 100 nm
in diameter, spherical and the outer layer is a lipid envelope displaying viral glycoproteins.
Each particle contains two copies of the linear viral RNA genome (approximately 10 kb),
(Figure 1) which contains three essential genes, gag, pol and env. The pol gene encodes
three viral enzymes: the protease, reverse transcriptase, and integrase. The gag gene
encodes the structural proteins: the capsid, matrix, and nucleocapsid. Proteins are
generated by proteolytic cleavage of the gag-pol precursor. The env gene encodes the
envelope glycoproteins of the virus. After the retrovirus enters the target cell, the viral
genome is converted into the double-stranded DNA form by reverse transcriptase (RT).
Proviral genome is then integrated into the genome of the target cell by the integrase. Viral
long terminal repeats (LTRs) are important for the initiation of viral DNA synthesis,
integration and regulation of viral transcription (Goff.S.P., 2001).
Figure 1. Structure of a Retroviral particle
Retroviral vectors can be easily engineered to encode an expression cassette. The
ability of the retrovirus to integrate and achieve long term expression has made them
attractive tools for gene transfer protocols, including functional studies (Taxman 2006),
expression of heterologous proteins, silencing of genes (Rubinson 2003, Singer 200 and
Sui 2002) and
gene therapy (Tolstoshev, 1992). As a safety issue, it is possible to
generate defective retroviral particles that wouldn’t replicate in the target cell. The genes
associated with replication are removed from the viral vector genome creating a defective
6
retrovirus which can efficiently transduce a target cell but lack information to replicate
(Naviaux et al, 1996; Bajgelman et al., 2003). Considering its utilization, retroviral vectors
has been used in the 20% of clinical trials in gene therapy. (Figure 2)
Figure 2. Vectors used in gene therapy clinical trials
Lentiviral vectors : improving gene transfer efficiency
Human lentivirus are members of the retrovirus family and derive from human
immunodeficiency virus (HIV). Concerning safety issues, these vectors keep less than 5%
of the parental genome, and less than 25% of the genome is incorporated into packaging
constructs, which minimizes the possibility of the generation of recombinant replicationcompetent HIV. Biosafety has been further increased by the development of selfinactivating (SIN) vectors that contain deletions of the regulatory elements in the 3’ long
terminal repeat sequence (LTR), to avoid the possibility of generation of replication
competent retrovirus (RCR) particles in the target cell (Thomas, Ehrhardt, & Kay, 2003).
Lentiviral vectors usually have a posttranscriptional regulatory element called WPRE that
enhanced mRNA stability and increase viral expression (Zufferey et al., 1999). With this
features they can transduce both dividing and quiescent cells, and allow to establish
permanent cell lines (Naldini et al, 1996, Strauss et al., 2006). In contrast with murine
retroviral vectors, lentivirus are less susceptible to methylation and are suitable to
generate transgenic animals (Tiscornia et al., 2003, Ikawa et al., 2003, Bressan et al,
2011).
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The Lentivirus preparation can be transiently produced by co-transfection of plasmids
encoding viral genome and packaging plasmids. After transfection viral particles are
released in the supernatant and can be purified (Figure 3).
Figure 3 Lentiviral production and transduction of a target cell. The lentivirus can be
transiently produced by co-transfection of viral plasmids that encode viral genome,
reverse transcriptase, structural proteins and envelope glycoprotein (1-3). Viral
particles that are assembled into the cell are released outside the cell and can be
harvested in the supernatant (4-6). This viral preparation contain viable particles
that lack packaging information and can be used to transduce a target cell, which
will express the gene of interest (7-13).
Evaluating transduction efficiency and concentration of a viral preparation
A critical issue regarding virus production is to determine the infectivity and
concentration of viral preparation. A number of methods to evaluate lentiviral titer have
been described. Titration can be performed quantifying viral antigens as the p24 by Elisa,
or using a biological method that allows an accurate detection of functional particles;
based on antibiotic selection, detection of reporter genes by flow cytometry or quantifying
8
transducing units using qPCR (Geraerts et al, 2006). Titration methods have advantages
and limitations that should be considered. Next we are going to review some methods
commonly used for lenviral titration:
Non functional methods
P24 antigen ELISA:
This test is directed toward the capsid protein p24. The approach is generally used
to determine the number of ‘physical’ particles (pp), And is based on the estimation that a
HIV core particle is composed of two thousands capsid proteins, (Farson, 2001). In this
way 1 fg of p24 represents around 12 pp. From this calculation, it is possible to estimate
viral concentration for any HIV-1-based lentiviral preparation. The estimation is routinely
employed for quality control validation of the different batches being produced.
Nevertheless, this technique can overestimate the functional vector titer because the p24
protein pool that is quantified includes a variable amount of free p24 that is originated from
non-functional vector particles.
Reverse transcriptase activity
Assessment of viral titer can be estimated performing a quantitative assay to
determine reverse transcriptase activity using an Elisa method. As seen for the p24 assay,
this non functional assay can mislead viral titer (Bukrinsky, et al.1993).
Determination of the genomic RNA concentration
Determination of the genomic RNA concentration in vector preparations can be
done by semi-quantitative northern blotting or dot blot analysis, however these techniques
lack accuracy to quantify the viral RNA content and viral titer. Moreover, RNA titers will
also assess defective particles that can mislead viral titer (Forghani 1991).
Functional methods
Lentiviral vector titration from transgene protein expression
A more accurate, functional titer is determined by transduction of cells following
limiting dilution of viral preparations and subsequent evaluation of reporter protein activity,
(e.g. betagalactosidase positive cells) or by assessment of the number of colony forming
units following antibiotic selection. Depending on the type of reporter used, lentiviral
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transducing units are defined in several ways. Colony forming units are generally used for
drug selection genes. (Metharom 2000).
The term of transducing unit used in the context of lentiviral vector gene transfer
has been routinely employed, especially for the expression of living color and lacZ genes.
(D’Costa 2003)
Flow cytometry
Flow cytometry (FC) allows quantifying cell populations based on fluorescence. A
viral vector encoding a fluorescent reporter gene, as GFP, can be titrated against non
transduced cells to estimate the number of transduced cells using a known volume of viral
preparation. Therefore is possible to extrapolate viral concentration to calculate viral titer.
As described in this work, flow cytometry may have some issues regarding viral dilution,
time of viral incubation before titration and the cell line that is chosen to the assay. Another
limitation of this technique is associated to the reporter gene, which should be a
fluorescent reporter gene (as GFP, dsRed, etc.), or even another gene which product
could be labeled by a fluorescent antibody.
Quantitative Polymerase chain reaction
An accurate method to quantify functional particles can be accomplished by
determination of the integrated proviral DNA copies per cell by Quantitative Polymerase
chain reaction (qPCR).
The qPCR is a real time PCR that allows monitoring reaction as it
progresses. The principle of detection is binding of a reporter molecule to double stranded
amplicon, or hybridization of a fluorescent-labeled probe to one amplicon strand. The
reaction requires minimal amounts of nucleic acid as template, there is no needing for the
post-PCR processing and the quantization of the end product is accurate. These
advantages of the fluorescence-based qPCR technique have completely revolutionized
the approach to PCR-based quantification of DNA and RNA. Real-time assays are now
easy to perform, have high sensitivity, more specificity, and provide scope for automation.
(Delenda, C ., Gaillard, 2005).
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Titration analyzing gene expression may provide the most useful data for predicting
the performance of vector supernatants in various cell types; however, two studies have
shown that this method likely underestimates the number of vector genomes transferred
into target cells, because variability in gene expression will lead some marked cells to be
counted as unmarked (Sastry et al., 2002; Lizée et al., 2003). Consequently, a more
accurate determination of the total number of infectious units may be achieved by
analyzing gene transfer by quantitative polymerase chain reaction (PCR). (Logan et al.,
2004).
11
Objectives
The main target of this work is to standardize a functional assay to titrate virus using flow
cytometry.
Specific aims
1. Determine a linear range for viral dilutions to avoid multiple integrations
underestimating titer, and also to establish a cut-off for the lower percentage of
transduction labeling to avoid overestimate titer.
2. Investigate the importance of incubation time after transduction before submitting
cells to flow analysis.
3. Evaluating titration using two different cell lines.
12
Material and methods
Cell lines and culture conditions
The murine cell line derived from fibroblasts NIH3T3 and the human HT 1080
derived from lung tumor fibrosarcom cell were maintained in DMEM (Gibco-BRL, Grand
Island, NY, USA) plus 10% Fetal Bovine Serum (HyClone – USA), at 37°C, 5% CO2.
Virus production
Virus was transiently produced by Viral vector laboratory – LNBio, co-transfecting
packaging cell line with a viral plasmid encoding the GFP reporter and packaging plasmids
encoding structural proteins and vesicular stomatitis vírus glycoprotein (VSV-G) envelope.
The lentiviral platform used was a second generation, in which the packaging
information is split in two different plasmids. The viral plasmid is self-inactivating (SIN) and
also has a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to
increase virus expression. The virus produced is defective because it lacks information to
replicate in the target cell, and can’t be packed by other wild type virus due the SIN-LTR.
Virus transduction
Virus transduction: Cells were seeded and transduced the next day with viral
supernatant diluted 1:100 in complete media plus 8ug/ml of polybrene. The cells were then
incubated for 24h, harvested or were changed media and harvested next day. To
harvesting for flow citometry, cells were trypsinized and resuspended in 1ml of 1X
phosphate buffer saline (PBS).
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Flow cytometry
The cell suspension was analyzed using a FACScalibur (Becton-Dickenson, CA,
USA) flow cytometer. The percentage of EGFP-positive cells is used to extrapolate the
number of infected cells. The dilution factor is then applied to arrive at the number of green
fluorescence units per milliliter (gfu ml-1) of virus supernatant. According to the equation:
100
100
1000
#
!"
P: percentage of GFP(+) cells, Negative: background of negative control, #cells: number
of cells on the transduction day, vol: volume of virus (ul), dilution of virus stock.
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Results and discussion
All the experiments were performed using aliquotes of purified FUGW virus from the
same batch , lot#071211, produced by Viral vector lab - LNBio. Transduction was done as
described in material and methods, altering some parameters as necessary, and indicated
in the experiments.
Determination of linear range for viral dilutions
Is common sense that using more than one particle of virus per cell should
underestimate viral titer, because flow cytometry will detect the percentage of transduced
cells. On the other hand, it is not clear what should be the minimal amount of viral particles
to assure transducing cells with one copy of virus per cell.
To investigate the relationship between transduction and amount of virus we
designed an experiment of titration, using experiment number (3), with cell line NIH3T3,
number of cells 4x104 and incubation time of 48 hours, adding different volumes of virus
per cell. As seen in figure 4, we have found linearity considering percentage of
transduction and viral volume from 1 to 25 ul of viral preparation. Using 25ul yielded 46%
of transduced cells, however using twice this volume (50ul) the transduction efficiency did
not follow the same pattern. In addition, using 50ul the intensity of signal increased,
suggesting the possibility of multiple viral integration.
According our experience and experimental data, we also notice that we could have
an error up to 20% between duplicates. Because of this, to increase precision we
considered to work with percentages of GFP positive cells, higher than 10%. So, it was
possible to determine a linear range for the titration curve, choosing 5 ul of our 1:100
diluted viral stock (lot#071211) as a standard, to compare titration between different
experiments. This because 5ul is the minimal virus volume that allows a good percentage
of GFP positive cells close to one particle of virus per cell.
15
Figure 4 – Determining linear range for flow cytometry-based titration using experiment
number (3), with cell line NIH3T3, number of cells 4x104 and incubation time of 48 hours.
(A) Raw data: Percentage of GFP positive cells and intensity obtained by flow cytometry.
(B) Graph representing the percentage of transduction versus viral volumes and (C) table
summarizing experimental condition, transduction efficiency and calculated titer
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Level of reporter expression is crucial for titration accuracy
In this assay we investigated the sensitivity of our titration method regarding
detection of the GFP reporter gene. The experiment consisted in evaluate detection of
GFP in two different experiments using the same amount of virus. The first experiment
was incubated 24h after transduction, and the second experiment was incubated 48h after
transduction. As seen in Figure 5, the viral titer is underestimated 90% when incubation
time was 24h versus 48h.
48 hs incubation time NIH3t3
24 hs incubation time NIH3t3
Figure 5- Titration accuracy depends on gene reporter expression. The figure shows two
titration experiments that were performed using the same batch of virus. As seen above,
incubating 24h yields only 10% of positive cells that could be detected by flow, compared
with 48h.
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Evaluating titration using two different cell lines.
The last parameter analyzed was to investigate the influence of cell line
transduction efficiency in viral titration. Is very well known that, there are cell lines more
susceptible to viral transduction and other cell lines that are more difficult to transduce. In
this way we compared the viral titer estimated using a very well described standard cell
line NIH 3T3 versus the HT1080 that is easily transduced with viruses. The first cell line is
a murine cell line derived from fibroblasts called NIH 3T3, and the second cell line is the
human HT 1080 derived from lung tumor fibrosarcom. The transduction was performed
using different viral dilution per cell line, and incubating 48h. As seen in Figure 6 the cell
line NIH 3T3 has shown a better transduction efficiency, and this cell line also yielded a
better titer. It is important to observe both cell lines yielded titers in the same magnitude,
what is consistent for these cell lines that are easily transduced with virus from ours
system. Figure number 6
NIH 3t3 (48 Hs INCUBATION)
Ht1080 (48 Hs INCUBATION)
Figure 6 - Comparison transduction efficiency by flow citometry. NIH 3T3 and HT1080 were
transduced using the indiceted amount of virus from the same batch. Cells were incubated
48h for transduction.
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Conclusions
The infectivity and concentration of a lentivirus preparation can be evaluated using a
suitable titration method. Here in we optimized a flow cytometry titration protocol which
requires a GFP reporter gene cloned in the viral vector. This method is based on detection
of transduced cells that are expressing the reporter gene. The analysis of our experimental
data indicated some aspects that have to be considered to optimize titration:
1. There is a linear range for flow cytometry detection. Flow cytometry can detect
percentage of transduced cells and it is important to analyze several dilutions to
verify a linearity between volume of viral preparation and percentage of transduced
cells. In our data we found that 5ul of volume virus is the best amount of virus to
have a good percentage of GFP positive cells avoiding multiple viral integrations.
2. The time of incubation is crucial for GFP detection. Considering a 100% GFPexpressing cells population, only 10% of GFP- transduced cells are detected within
24h.
3. Tranduction efficiency depends on the susceptibility of the cell line, and can
interfere in viral titer. We compared titration of a standard cell line, NIH 3T3, versus
HT1080, using the same batch of virus and same incubation time (The NIH 3T3 is
being used by LVV to titrate viral preparations). Both cell lines exhibited a good
level of reporter gene expression in the same magnitude, however, the NIH 3T3 cell
line shows a better transduction efficiency, and also yielded a better titer.
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Additional experiments
During the period I worked in the Viral Vector Lab - LNBio, besides performing
experiments with flow cytometry I had the opportunity to be introduced to other activities
that are routine in this lab. I spend a few weeks being trained in molecular biology. I
prepared plasmids, performed restriction maps and started cloning vectors that will be
used in the lab. Also I had the opportunity to start the standardization of another titration
method based on qPCR. This method has the advantage of detecting virus that lacks a
reporter gene for flow cytometry.
Next I summarize some data produced during this period that would be used as
preliminary data for qPCR titration.
Preliminary assays to Standardize of qPCR titration
Objective
The main purpose of this experiment was to validate oligos designed for titration of
targets, as the WPRE and Neo cDNA.
Material and Methods
The qPCR was done using the syber green reagent (Applied Biosystems – USA)
and custom primers developed in the lab. The standard curves were done using plasmid
dilutions, encoding target cDNAs, as template.
Conditions of qPCR for Neomycin resistance gene and WPRE element
qPCR reactions were prepared in 96-wells provided by applied Biosystems, with
10µl of syber green, 0,08 µl of primers (100um) (forward and reverse) for neomycin and
WPRE element, and 4,92 µl of miliq water, DNA amplification was carried out using an
Applied Biosystems 7500 Real-Time PCR Sequence detection system using cycling
conditions of 95°C 10 minutes, 40 times: 95°C 15 se conds and 60°C 1 minute, 95°C 15
seconds 60°C 20 seconds and 95°C 15 seconds.
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Results
Standard curve for WPRE
For the standard curve were prepared serial dilutions that were made in triplicates,
starting from 106 to 100 copies of plasmid template encoding desired target. The results
obtained show a R2 of 0.9827, which indicates a good correlation between the different
dilution points (Figure 7).
6
0
Figure 7 Standard curve for WPRE with dilutions (10 -10 )
Standard curve for Neomycin
For the standard curve were prepared serial dilutions that were made made in
triplicates, starting from 106 to 100 copies of plasmid template encoding desired target. The
results obtained show a R2 of 0.9626, which indicates a good correlation between different
dilutions. (Figure 8).
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Figure 8 Standard curve of Neomycin
Testing titration of transduced cells
The aim of this assay was to perform a quantitative detection of virus from
transduced cells. The first step was to establish a cell line expressing an internal control
that could be used to normalize the absolute quantization. Therefore we transduced NIH3T3 with a diluted viral preparation of pCL virus which encodes neomycin resistance gene.
After transduction cells were selected with G418, establishing a pool of resistant cells that
should have one copy of neomycin gene per cell. Next step was to transduce the
neomycin resistant cells with a FUGW virus, which encodes GFP and also has the WPRE
element. In this way we performed two different transductions: sample#1 was transduced
with 50 ul of virus and sample#2 was transduced with 25 ul of virus. Cells were analysed
by flow cytometry to confirm the expected virus expression and then the genomic DNA
(gDNA) was isolated, quantified and then we used 100ng as template for qPCR.
As seen in Figure 9, our internal control (neomycin) was detected around 294
copies/100ng of gDNA for sample#1 and a very similar level was detected for sample#2:
224 copies/100ng of gDNA. This data is consistent because samples 1 and 2 derives from
the same batch of neomycin expressing cells and should have the same copy number of
neo gene.
On the other hand, quantitation of WPRE wasn’t consistent, and should be repeated
for further experiments (Figure 9)
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Figure 9. Comparison of the qPCR results between Neomycin and WPRE
Conclusion of qPCR titration assay
The qPCR should provide an accurate method for viral titration. In this experiment
we checked the feasibility of the method and more experiments should be performed to
optimize conditions.
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