Design and Production of
Lentiviral vectors (LV) can govern the efficient
delivery and stable integration of transgenes both in
and in vivo
, can transduce a wide range of targets,
including stem cells, and can be used for generating
transgenic animals from several species. Lentiviral
vector-mediated gene transfer results in the ubiquitous,
tissue-specific, and/or regulated expression of
these transgenes, depending on the promoter contained
in the vector. Finally, this gene delivery system
can mediate the knockdown of endogenous genes by
RNA interference via the polymerase III promoterdriven
production of small hairpin RNAs.
The potential of lentiviral vectors was first revealed
in 1996 through the demonstration that they could
transduce neurons in vivo
(Naldini et al.
, 1996). Since
then, many improvements have been brought to
achieve high levels of efficiency and biosafety. The
principle, however, remains the same and consists of
building replication-defective recombinant chimeric
lentiviral particles from three different components:
the genomic RNA, the internal structural and enzymatic
proteins, and the envelope glycoprotein. A
schematic diagram of the evolution of human immunodeficiency
virus (HIV)-based vectors is represented
in Fig. 1. The genomic RNA contains all cis
-acting sequences, whereas packaging plasmids contain all the trans
-acting proteins necessary for adequate transcription,
packaging, reverse transcription, and integration.
|FIGURE 1 Evolution in the design of HIV-l-based LV vectors. HIV-l-based LV vectors are derived from
wild-type HIV-1 by dissociation of the trans-acting components (blue boxes, above HIV genome) coding for
structural and accessory proteins and the cis-acting sequences required for packaging and reverse transcription
of the genomic RNA (golden boxes, below HIV genome). Sequences added between two vector
versions are in red. CMV, human cytomegalovirus immediate-early promoter; RRE, rev-responsive element;
RSV, Rous sarcoma promoter; polyA, polyadenylation site; U3-R-U5, HIV-1 LTR: SD, major splice donor; psi,
HIV-1 packaging signal; cPPT, central polypurine tract; SA, splice acceptor; dPPT, distal (3') polypurine tract;
Prom, promoter of the internal expression cassette; TG, transgene of the internal expression cassette; ΔU3,
self-inactivating deletion of the U3 part of the HIV-1 LTR; WPRE, woodchuck hepatitis virus posttranscriptional
Although various envelope proteins can efficiently
pseudotype LV particles (Sandrin et al.
, 2002), the G
protein of the vesicular stomatitis virus (VSV) is the
most widely used. The main reasons for this choice
are that the VSV envelope (1) allows for high titers
achieved in unconcentrated supernatants; (2) provides
an extremely wide range for the transduction of target
cells (virtually all mammalian cells of any tissue tested
so far can be transduced by VSV-G pseudotyped
LVs); (3) is very robust, allowing for concentration by
ultracentrifugation; and (4) has a good resistance to
B. Core and Enzymatic Components
The first-generation lentiviral vectors were manufactured
using a packaging system that comprised all
HIV genes but the envelope (Naldini et al.
, 1996). In a
so-called second-generation system, five of the nine
HIV-1 genes were eliminated, leaving the gag
reading frames, which encode for the structural
and enzymatic components of the virion, respectively,
and the tat and rev genes, fulfilling transcriptional and
posttranscriptional functions (Zufferey et al.
Sensitive tests have so far failed to detect replicationcompetent
recombinants (RCRs) with this system. This good safety record, combined with its high efficiency
and ease of use, explains why the second-generation
lentiviral vector packaging system is utilized for most
experimental purposes. In a third-generation system,
geared toward clinical applications, only gag, pol,
genes are still present, using a chimeric 5' long
terminal repeat (LTR) to ensure transcription in the
absence of Tat (see later).
C. Genomic Vector
The genetic information contained in the vector
genome is the only one transferred to the target cells.
Early genomic vectors were composed of the following
components: the 5' LTR, the major splice donor, the
packaging signal (encompassing the 5' part of the gag
gene), the Rev-responsive element (RRE), the envelope splice acceptor, the internal expression cassette
containing the transgene, and the 3' LTR. In the latest
generations, several improvements have been introduced.
The woodchuck hepatitis virus posttranscriptional
regulatory element (WPRE) has been added to
increase the overall levels of transcripts in both producer
and target cells, hence increasing titers and transgene
expression (Zufferey et al.
, 1999). The central
polypurine tract of HIV has also been added back in
the central portion of the genome of the transgene RNA
(Follenzi et al.
, 2000; Zennou et al.
, 2000). This increases
titers in some targets. The 3' LTR has been deleted in
the U3 region to remove all transcriptionally active
sequences, creating the so-called self-inactivating (SIN)
LTR (Zufferey et al.
, 1998). Finally, chimeric 5' LTRs
have been constructed in order to render the LV promoter
Tat independent. This has been achieved by
replacing the U3 region of the 5' LTR with either the
CMV enhancer (CCL LTR) or the corresponding Rous
sarcoma virus (RSV) U3 sequence (RRL LTR) (Dull et
al., 1998). Vectors containing such promoters can be
produced at high titers in the absence of the Tat HIV
transactivator. However, the Rev dependence of these
third-generation LV has been maintained in order to
maximize the number of recombination events that
would be necessary to generate an RCR (Fig. 2).
III. MATERIALS AND REAGENTS
|FIGURE 2 Vector for RNA interference. Small hairpin (interfering)
RNAs (shRNA or siRNA) are expressed from a polymerase III
promoter as described (Brummelkamp et al., 2002). In this example,
the expression cassette is placed in the U3 region of the 3' LTR.
Because of the modalities of reverse transcription, two copies of the
siRNA inducing module will be present in the integrated provirus,
facilitating high levels of production. For convenience, a transgene
can be placed in the same vector, downstream of an internal
- 293T and HeLa cell lines (ATCC) (www.atcc.org)
- Chemicals (Sigma-Fluka) (www.sigmaaldrich.com)
- Cell culture media and additives (GIBCO-BRL-Life
Technologies) (www.gibcobrl.com) and CellGenix
Technologie Transfer GmbH, Germany (www.
- Plastics for tissue cultures, flow cytometer (BD
- DNA purification kits (Qiagen) (wwwl.qiagen.com)
and Genomed (www.genomed-dna.com)
- Centrifuges (Sorvall) (www.sorvall.com)
- Filters (Millipore) (www.millipore.com)
- Ultracenrifuge tubes (Beckman) (www.beckman.com)
- Sequence detector ABI7700 plus ABI SDS software
for analysis, optical reaction plates and caps
for QPCR (Applied Biosystems) (http://www.
- QPCR reaction mixes and probes (Eurogentech)
- HIV-1 p24 antigen capture assay (AIDS Vaccine
Program) (Frederick, MD. email: schadent@mail.
The production of LV can be achieved by transient
transfection of the plasmid set into 293T cells by the
calcium phosphate method or from stable producer
cell lines. Although proof of principle for the latter
approach has been provided (Klages et al.
, 2000), it still
suffers from limitations, e.g., for the production of SIN
vectors. Unless very large amounts of a same vector
are needed on a regular basis, transient production is
still the method of choice for research purposes.
Cells (293T/17 from ATCC Cat. No. SD-3515) are
probably the most critical factor for good titers. They
need to be passaged every 2-3 days, as they start to
form clumps that cannot be dissociated with one
round of trypsin.
- 0.5M CaCl2: Dissolve 36.75 g of CaCl2 and 2H2O (MW 147) (SigmaUltra Cat. No. C5080) into 500ml of
H2O (distilled or double distilled). Store at -70°C in
50-ml aliquots. Once thawed, the CaCl2 solution can be
kept at 4°C for several weeks without observing a significant
change in the transfection efficiency.
- 2x HeBS: Dissolve 16.36g of NaCl (MW 58.44)
(SigmaUltra Cat. No. S7653 (0.28M final), 11.9g of HEPES (MW 238.3) (SigmaUltra Cat. No. H7523)
(0.05M final), and 0.213g of Na2HPO4, anhydrous
(MW 142) (SigmaUltra Cat. No. S7907) (1.5mM final)
into 800ml of H2O (distilled or double distilled).
Adjust pH to 7.00 with 10M NaOH. Be careful, as
obtaining a proper pH is very important. Below pH
6.95, the precipitate will not form, above pH 7.05, the
precipitate will be coarse and transfection efficiency
low. Then add H2O to 1000ml and make the final pH
adjustment. Store at -70°C in 50-ml aliquots. Once
thawed, the HeBS solution can be kept at 4°C for
several weeks without observing a significant change
in the transfection efficiency.
- Solution for mixing with plasmids: 50ml H2O distilled
or double-distilled, 1M HEPES, pH 7.3 (Gibco- BRL, Cat. No. 15630-056) 125µl (2.5mM final). We
have observed that the aspect and quality of the precipitates
can vary among batches of distilled water.
To circumvent this problem, we advise buffering
distilled water that is used to dilute the plasmids. A
final concentration of 2.5mM HEPES in the water
will help maintain a proper pH and will not compete
for the final pH with the HeBS, pH 7.00 (HEPES, pH
7.00, provided by HeBS is 25mM final, whereas
HEPES, pH 7.3, provided by water is 0.625M final).
Store at 4°C.
We use JetStar kits (Genomed GmbH, Germany) or
Qiagen kits (Qiagen, GmbH, Germany) to prepare
DNAs for transfection. In any case, the last step of the
DNA prep should be an additional precipitation with
ethanol (EtOH) and resuspension in 10mM
Cat. No. T6791) /1mM
Cat. No. E6758) (TE
10/1). Do not treat DNA with
phenol/chloroform as it may result in chemical alterations.
Also, to avoid salt coprecipitation, we do not
precipitate DNA below 20°C.
D. Transfection and Harvesting
|FIGURE 3 Phase-contrast photograph of 293T cells. 293T/17
are seeded the day before transfection at 1 to 3 million
culture dish. At the time of transfection, cells must
have the morphology
and density as shown here.
The day before transfection, detach the 293T cells
using trypsin/EDTA (Cat. No. 25300, GibcoBRL Life
Technologies). Seed the 293T cells at 1 to 3 millions
(cells must be approximately one-fourth to one-third
confluent on the day of transfection) per 10-cm culture
dishes (Cat. No. 353003, BD Biosciences) with 10ml of
D10 complete medium composed of DMEM (Cat. No.
41966, GibcoBRL Life Technologies) supplemented
with 10% fetal calf serum (FCS, Cat. No. 10099, Gibco-
BRL Life Technologies), 1% penicillin-streptomycin
(Cat. No. 15140, GibcoBRL Life Technologies), and 1% L
-glutamine (Cat. No. 25030, GibcoBRL Life Technologies)
On day 0, in the evening, make the precipitate
according to the recipes 1
(cf Table I for one 10-cm plate).
These recipes are the result of long optimizations in our laboratory
and in the laboratory of Luigi Naldini and discussions with
Adjust to 250µl with buffered water. Add 250µl of
. Mix well and add this mix, dropwise,
(one drop every other second), on 500µl of HeBS
2×, while vortexing at maximum speed. Let stand still
for 20 to 30min minimum (40 min max) on the bench.
This time is critical for optimal formation of the precipitate.
Excessive incubation times may induce the
formation of a coarse precipitate, which is detrimental
to transfection efficiency. A too short incubation will
not induce the formation of a significant amount of
precipitate, which will have no chance to develop
further once diluted in the 10ml of culture medium.
Also, it is advisory to incubate the solutions at room
temperature or 37°C before mixing to standardize the
precipitate characteristics. Add the precipitate slowly,
dropwise on the cell monolayer. The plate should then
be shaken gently but not stirred or all the precipitate
will be in the center. At this point, the precipitate
may not be visible, for it is too fine and will take
several hours to sediment on the cells. If the precipitate
is readily visible, it is too coarse and the transfection
will most likely be less efficient.
On day 1 in the morning, check the cells. At
this point, a very fine and sandy precipitate all over
the plate should be seen, except on the cells and in
their vicinity, possibly because cells absorb the
CaPO4/DNA precipitate. Discard the medium after
gentle, but firm, stirring (to eliminate the maximal
amount of precipitate) and replace it with 10-15ml of
: The medium must be prewarmed at 37°C because the 293T are very sensitive to thermal shock
and can shrink and detach. Also, the medium must be
added very gently to the cells. Even so, it is difficult
not to make a hole in the monolayer.
On day 2, harvest the supernatant and replace
with 10-15ml of medium as the day before. Spin
the supernatant at 2500rpm for 10min at 4°C [in a
tabletop centrifuge such as a Multifuge 3SR (Sorvall)],
filter through a 0.45-µm PVDF filter (such as Millex-
Durapore, Cat. No. SLHV 033, Millipore), and store
On day 3, harvest the supernatant, spin at 2500 rpm
for 10min at 4°C, filter through a 0.45-µm filter, and
pool with the supernatant of day 2. At this point, the
pool of supernatants must contain approximately 106
transducing units (TU) per milliliter (as titered on
HeLa cells, see later).
V. CONCENTRATION AND
If a more concentrated lentivector suspension is
required, the vector preparation needs to be concentrated.
For concentration, we use Beckman Conical
tubes (Cat. No. 358126, Beckman-Coulter
) in a Surespin
630 rotor and a Discovery 90SE ultracentrifuge
(Sorvall). Put 4-5 ml of 20% sucrose (SigmaUltra, Cat.
No. S7903) in water at the bottom of the tube and
fill up to the top with filtered supernatant, spin at
2600 rpm for 90 min and discard gently the supernatant
by inversion. Let the tube dry inverted and resuspend
the pellet (not always visible) with complete medium
or serum-free medium, such as CellGro stem cell
growth medium (Cat. No. 2001, CellGenix Technologie Transfer GmbH, Germany) if the subsequent experiments
require the absence of serum.
The use of protein-containing medium is preferable
over PBS to resuspend the viral pellet for two reasons:
(1) the presence of proteins will stabilize the viral particles
and (2) the surfactant effect of proteins will help
redissolve the pellet.
Titers of viruses in general and lentivectors in particular
critically depend on the method and cells used
for titration. The quantification of vector particles
capable of achieving every step from cell binding to
expression of the transgene depends on both vector
and cell characteristics. First, the cell used as the target
must be readily permissive to all steps from viral entry
to integration of the vector genetic cargo. Second, the
expression of the foreign gene must be monitored
easily and rapidly reach levels sufficient for reliable
quantification. Early vectors had the LacZ bacterial
gene as the reporter under the control of the CMV promoter
(Naldini et al.
, 1996). Current vectors now have
the green fluorescent protein (GFP) gene as a reporter
under the control of promoters that are active in most
primary cells (Salmon et al.
, 2000). Measured titers can
also vary with the conditions used for titration, i.e.,
volume of sample during vector-cell incubation, time
of vector-cell incubation, and number of cells used. For
several years now, we have been using HeLa cells as
target. These cells are stable, easy to grow, and 100%
susceptible to transduction by VSV-G-pseudotyped
LVs. There are also now used commonly by many laboratories,
which helps in comparing titers between
A. Titration of Vectors in HeLa Cells
This method can only be used to titer stocks of
vectors that carry a transgene that is easily monitored
by FACS (such as GFP, any living colors, or any
membrane protein that can be detected by flow cytometry)
and whose expression is governed by a promoter
that is active in HeLa cells (tissue-specific promotercontaining
vector must be functionally assayed in specific
cells), and titered by quantitative polymerase
chain reaction (QPCR) in HeLa cells (see later).
We describe here the titration of a PGK-GFP vector.
On day 0, seed Hela cells (Cat. No. CCL-2, ATCC) at
100k cells per well in a MW6 plate (Cat. No. 353224,
BD Biosciences) in D10 complete medium. On day 1,
put 500, 50, or 5 µl of the vector suspension (either pure
from unconcentrated supernatants or diluted if it
comes from a concentrated stock) in three independent
wells. On day 2, remove the supernatant and
replace with 2ml of fresh D10. On days 4 to 5, wash
the cells with 2 ml of PBS without calcium-magnesium
(Cat. No. 14190, Gibco-BRL Biosciences), detach them
with 250µl of trypsin/EDTA (Cat. No. 25300, Gibco-
BRL Life Technologies) for 1 min at 37°C, add 250 µl of
2% (w/v) formaldehyde (Cat. No. F8775, Sigma) in
PBS without calcium-magnesium (to fix the cells, inactivate
the trypsin and the vector particles), resuspend
thoroughly, and analyze them for GFP expression
using a flow cytometer (FACScan, Becton-Dickinson).
A reliable measure of the fraction of GFP+ cells
relies on the level of GFP expression. In the example
shown in Fig. 4, GFP-positive and GFP-negative cells
can be readily discriminated when GFP is expressed
from a human PGK promoter and allowed to accumulate
in cells for 4 days. A marker can then be set to
measure the fraction of transduced versus total cells.
In a typical titration experiment, only dilutions
yielding 1-20% GFP positive should be considered for
titer calculations. Below 1%, the FACS may not be
accurate enough to reliably determine the number
of GFP-positive cells. Above 20%, the chance for each
GFP-positive target cell to be transduced twice
increases significantly, resulting in an underestimation
of the number of transducing particles. Once the
appropriate dilution is chosen, apply the following
titer (HeLa-transducing units/ml) = 100,000 (target
HeLa cells) × (% of GFP-positive cells/100)/volume
of supernatant (in milliliters).
B. Total Vector Concentration Using
|FIGURE 4 A representative FACS analysis of HeLa cells used for titration of GFP-coding LV. HeLa cells (105)
were incubated with various volumes of a supernatant containing a LV expressing GFP under the control of
the human PGK promoter [pRRLSIN.cPPT.PGK.GFP.WPRE (Follenzi et al., 2000)] as described in the text.
After 4 days, cells were detached, fixed, and analyzed by FACS for GFP fluorescence (x axis, four-decade log
scale, FL1) versus number of cells (y axis, linear scale). The percentage of GFP-expressing cells was measured
by placing a marker discriminating between GFP-negative (mean of fluorescence intensity 3-4) and GFPpositive
(mean of fluorescence intensity 200) cells.
Determination of total particle concentration is
important to monitor the efficiency of vector production
and packaging. One must keep in mind,
however, that a high concentration of pelletable p24
viral capsid antigen can be measured from vector particles
not containing any genomic RNA and/or devoid
of envelope protein. Such an assay can thus not replace
a transduction assay as described earlier and later. We
currently use the p24 antigen capture assay provided
by the AIDS vaccine program (Frederick, MD; email:
email@example.com). The minimal p24 concentration
detected by this assay is 150pg/ml. HIV-1 or
vector-containing supernatants are lysed by adding
Triton X-100 (SigmaUltra Cat. No. T9284) to a final
concentration of 1%. Lysed samples can be analyzed
just after or stored at -20°C. Following the instructions
provided by the manufacturer and using dilutions
comprised between 1 / 10 and 1 / 10,000, we find that
most of our vector preparations have a p24 concentration comprised between 100ng/ml and 1µg/ml.
Given that most vector preparations have titers
ranging from 5 × 105
Hela-TU/ml, this implies
that the relative titer of our LV stocks is comprised
between 1 and 20 Hela-TU/pg of p24. Of course, these
results may vary depending on the packaging efficiency
of a given transfer vector.
C. Titration of Vectors in HeLa Cells by
In the case of vectors coding for genes that cannot
be detected readily by FACS or if the promoter is not
active in HeLa cells, an alternative method is to
measure the number of copies of LV stably integrated
in HeLa target cells, after transduction as described
earlier for GFP vectors. This assay, however, only
measures the number of LV copies integrated in the target
cell genome. The overall functionality of the vector
must be tested at least once in cells in which the
promoter is active and/or with appropriate techniques
to detect the expression of the transgene product.
The QPCR assay proceeds as follows using a 7700
sequence detector (Applied Biosystems). HeLa cells
are transduced as described earlier, but instead of
being detached by trypsin, they are lysed with 200µl
of lysis buffer in the plate and DNA is extracted using
a DNAeasy kit (Qiagen GmbH, Germany). Then, 1-
2 µl of 200 µl total of DNA solution is analyzed for the
copy number of HIV sequences using the following
real-time PCR protocol.
Always use filter tips. Mix for each sample and distribute
in duplicate in a 96-well optical reaction plate
(Cat. No. 4306737, Applied Biosystems): 12.5 µl 2× mix
(Cat. No. RT-QP2X-03 Eurogentech, Belgium), 2.5 µl 10× oligonucleotide mix 1, 2.5 µl 10× oligonucleotide mix 2,
1-2 µl DNA sample, and up to 25 µl final with H2
with optical caps (Cat. No. N801-0935, Applied Biosystems).
Run a program appropriate for the probes,
depending on the fluochrome used (FAM, VIC, TET, etc).
: 10× oligonucleotide mixes are 1 µM
of each primer in water. Stocks of probes and
primers usually come lyophilyzed and are stored at
in water. DNA ideally comes from 2 × 106
cells extracted and resuspended in 100µl (DNAeasy,
Oligonucleotides can be ordered online from
several companies such as Eurogentech or Sigma.
Oligonucleotides used to normalize for the amount
of genomic DNA are specific for the β-actin (BAC)
gene. The sequences are originally from a PE-Applied TAQMAN β-actin control reagent (Cat. No. 401846),
which has been discontinued.
BAC-P (probe, sense) human β-actin
5'-(TET or VIC)- ATGCCCTCCCCCATGCCATCCTGCGT -(TAMRA)-3'
BAC-F (forward primer
BAC-R (reverse primer
Oligonucleotides for the amplification of HIV-1-
derived vectors are specific for the 5' end of the gag
gene (GAG). This sequence is present in all HIV-1
vectors for it is part of the extended packaging signal.
GAG-P (probe, antisense) HIV gag
GAG-F (forward primer): GGAGCTAGAACGATTCGCAGTTA
GAG-R (reverse primer): GGTTGTAGCTGT CCCAGTATTTGTC
An example of amplification profiles of HIV
sequences in human DNA is given in Fig. 5 (as displayed
by the ABI Prism program, Applied Biosystems).
To analyze the amplification reaction, first set
the threshold where the amplification curve is the
steepest, both for the gene of interest (GAG-FAM, Fig.
5A) and for the internal control (BAC-VIC, Fig. 5B).
Then, export the results as a Microsoft Excel sheet
and draw a standard curve with standards of cells
containing 1, 0.1, 0.01, and 0.001 copy of HIV per cell
using the deltaCt values (Ct GAG minus Ct BAC). Ask
Excel to display the formula (exponential). Apply
the formula to unknown samples, it will give the HIV
copy number of the corresponding sample. The titer
of the supernatant can then be calculated as follows:
Titer (HeLa-transducing units/ml) = 100,000 (target
HeLa cells) × number of copy per cell of the
sample/volume of supernatant (in milliliters).
: It is advisory to run a dual titration (FACS plus
TAQMAN) using one GFP vector alongside the other
vectors for each set of QPCR titration. This will help in
comparing FACS titration with QPCR titration. A standard
curve of HIV DNA in human DNA can also be
made using a human cell line. In this case, keep in
mind that the DNA content of these cell lines is different
from the DNA content of normal primary human
diploid cells. Small adjustments can be made using the
table in Fig. 5D, which summarizes the chromosome
counts of HeLa and CEM cells as provided in the ATCC catalog and gives calculated DNA contents for
|FIGURE 5 A representative QPCR analysis used for titration of HIV-1-based LVs. DNA from 8E5 cells, a
CEM-derivative (Cat. No. CRL-8993, ATCC; which contains a single copy of a replication defective HIV-1
provirus), was diluted by ten-fold increments in DNA from CEM cells. A sample of each dilution was submitted
to QPCR amplification and monitoring using a Perkin-Elmer 7700 (Applied Byosystems) and sets of
primers and probes specific for HIV gag sequences (GAG-FAM, A) or β-actin sequences (BAC-VIC, B). Amplification
plots were displayed, and cycle threshold values (Ct) were set as described in the text. Values of GAG
Ct and BAC Ct were exported in an Excel worksheet to calculate ΔCt values (x axis, linear scale) and plot
them against copy number values (y axis, log scale) (C). The regression curve can then be used to calculate
GAG copy numbers (Y value) of unknown samples by applying the formula to ΔCt values (X values) of the
sample. (D) Calculated DNA contents of primary human cells, CEM, cells and HeLa cells based on the chromosome
contents (after ATCC catalog).
In the case of lack of transduction of a specific cell type
with a specific lentiviral vector, Fig. 6 helps in addressing
most of the problems that could account for it.
|FIGURE 6 Troubleshooting diagram for lentiviral vector production and transduction.
Lentivector are vehicles of choice for the transduction
of many primary cell targets, including neurons,
retinal cells, pancreatic islet cells, lymphocytes, and
hematopoietic stem cells. Although HIV-derived
vectors have been best characterized, gene delivery
systems have similarly been developed from other
lentiviruses, such as simian (Negre et al.
, 2000), feline (Curran et al.
, 2000), and bovine (Berkowitz et al.
immunodeficiency viruses and the equine infectious
anemia virus (Mitrophanous et al.
, 1999). These other
systems seem to have the same general properties as
their HIV counterpart, but in human cells, HIVderived
vectors appear to be more generally efficient,
illustrating the fact that a virus adapts to its cognate
target. From a biosafety standpoint, HIV-derived
vectors may be safer than their nonhuman virus homologues.
First, the genomic complexity of HIV is far
greater than that of most other lentiviruses, including
feline immunodeficiency virus and equine infectious
anemia virus, which each have only six genes instead
of the nine present in their human counterpart.
Because in all cases a minimum of three genes, gag, pol
, will likely be required for generating vector
particles efficiently, the multiply attenuated HIV-based
packaging system will be the farthest away from its
parental virus. In addition, past experience with
zoonoses teaches us that the pathogenicity of a given
organism is largely unpredictable when it is transferred
from its normal animal host into humans.
Finally, millions of individuals worldwide have been
screened for lentivirus-related diseases. No pathology
has been associated with massively deleted forms
of HIV-1; however, well-documented cases of longterm
clinical nonprogression have occurred in patients
infected with HIV-1 strains that carry genetic alterations
far more subtle than those introduced in the
third-generation HIV-1 packaging system.
Most recent developments of lentivector technology,
such as its application for transgenesis (Lois et al.
2002) and RNA interference (Brummelkamp et al.
2002), indicate that the exploitation of this formidable
tool is only beginning. Exciting times are ahead, in
both experimental and therapeutic arenas.
We thank Antonia Follenzi for helpful discussions
and Maciej Wiznerowicz for Fig. 2. Additional
resources, such as vector sequences, are available
at http://www.tronolab.unige.ch/. Downloading of
QPCR oligonucleotide sequences and Excel QPCR
calculation sheets can be made at http://www.
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