Leukemia (2002) 16, 563–569
 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00
www.nature.com/leu
REVIEW
Development of gene therapy for hematopoietic stem cells using lentiviral vectors
N-B Woods, A Ooka and S Karlsson
Lentiviral vectors are promising tools for the development of
gene therapy since they can transduce both quiescent and
dividing target cells. Lentiviral vectors may be particularly
promising gene delivery tools for hematopoietic stem cells
since these target cells tend to be quiescent and are therefore
difficult target cells for vectors that require dividing targets.
Human hematopoietic stem cells that can repopulate NOD/SCID
mice have been efficiently transduced using HIV-1-based lentiviral vectors and similar vectors can also transduce murine
hematopoietic stem cells. HIV-1 vectors that contain strong
general promoters can generate high levels of transgene
expression and very high expression levels can be generated
in erythroid cells in vivo using ␤-globin regulatory sequences
to control the expression of the transgene. Current lentiviral
vectors have a similar level of biosafety as oncoretroviral vectors and can therefore theoretically be used in clinical gene
therapy protocols. Future challenges include the generation of
lentiviral vectors that can express more than one transgene at
high levels and the generation of safe permanent packaging
cells for practical use in clinical gene therapy trials.
Leukemia (2002) 16, 563–569. DOI: 10.1038/sj/leu/2402447
Keywords: lentiviral vectors; hematopoietic stem cells; gene
therapy
Introduction
Hematopoietic stem cells (HSCs) are important targets for
gene therapy of genetic disorders of the blood system since
they have the ability to self-renew and are capable of differentiating into all blood lineages. Oncoretroviral vectors have
been the classical gene delivery vehicles for hematopoietic
cells, including hematopoietic stem cells. High efficiency
gene delivery of murine hematopoietic stem cells can be
accomplished using oncoretroviral vectors but efficient transduction of hematopoietic stem cells from humans or large outbred animals has been more problematic.1–4 The recently published successful gene therapy for severe combined immune
deficiency-X1 (SCID) due to ␥c chain deficiency has been a
breakthrough in the gene therapy field and provides proof that
this therapeutic modality works in practice.5 While these
advances are encouraging, problems remain. The success with
the SCID ␥c chain deficiency is based on in vivo selection of
early lymphoid progenitors following gene transfer of the ␥c
chain gene into autologous CD34+ bone marrow cells that are
subsequently transplanted back to the patient. All T cells and
NK cells from the treated patients contained the transgene
while only 1% of the myeloid progeny cells contained the
vector. This indicates that the gene transfer efficiency into
hematopoietic stem cells has been approximately 1% and that
the gene modified lymphoid progenitor cells will be selectively grown and differentiated in vivo upon stimulation by ILCorrespondence: S Karlsson, Molecular Medicine and Gene Therapy,
Lund University Hospital, Sölvegatan 17, 223 62 Lund, Sweden;
Fax: +46 46 222 0578
Received 12 October 2001; accepted 3 January 2002
2, IL-7 and IL-15 while the unmodified cells die due to the
lack of the cytokine receptors for IL-2, IL-4, IL-7, IL-9, IL-15,
of which the ␥c chain is a part. Since gene replacement of
most genetic defects in hematopoietic cells will not lead to in
vivo selection, gene transfer efficiencies that are substantially
higher than 1% will be needed for treatment of these
disorders.
Suboptimal gene transfer efficiency into human hematopoietic stem cells using oncoretroviral vectors is to a large extent
due to the quiescent nature of hematopoietic stem cells during
steady-state hematopoiesis. At any point in time, most hematopoietic stem cells are in G0 and to a lesser extent in G1,
while very few are in active cell cycle (G2/S/M).6–8 Since
active cell cycle is a requirement for transduction by oncoretroviral vectors,9,10 the hematopoietic cells need to be stimulated by growth factors prior to transduction. Active growth
stimulation ex vivo by three or more cytokines for 3–4 days
is common practice to allow enough time for active cycling
of primitive hematopoietic cells prior to and during oncoretroviral transduction.11–13 While it has been well documented
that this growth stimulation of early hematopoietic cells
improves gene transfer efficiency, it has detrimental effects on
engraftment during bone marrow transplantation. Hematopoietic progenitor and stem cells in G2/S/M engraft poorly compared to cells in G0 and G1.7,8,14,15 These difficulties have
focused attention on the use of lentiviral vectors for transduction of human hematopoietic stem cells (HSCs) in order to
achieve high gene transfer efficiency and efficient engraftment
of transduced hematopoietic stem cells.
Although many species-specific lentiviruses exist, most preclinical gene therapy experiments have been performed using
vectors based on the human immunodeficiency virus type-1
(HIV-1), and these represent the most advanced lentiviral vectors to date.16–21 Because the HIV-1 envelope protein binds
specifically to receptors on macrophages and T cells, another
envelope protein has to be used to allow the vector to enter
HSCs. Therefore, lentiviral vector particles pseudotyped with
the vesicular stomatitis virus glycoprotein (VSV-G) have been
used because they can enter a large variety of target cells due
to the ubiquity of the VSV-G phospholipid receptor, and they
can easily be concentrated by ultracentrifugation.16,22,23
Figure 1 shows how lentiviral vectors are generated using a
transient transfection packaging system where the transfer vector plasmid is transfected into 293-T cells with plasmids encoding the VSV-G gene and the HIV-1 gag, pol and rev genes.
This packaging system cannot create wild-type HIV-1 because
the genes for the HIV-1 env and the accessory protein genes
are lacking. The system described in Figure 1 is the third generation lentiviral vector system17 where the accessory protein
genes (essential for HIV-1 pathogenesis) are lacking and the
gag and pol genes on one hand and the rev gene on the other
are encoded by separate plasmids. Recently, a third generation packaging cell line for HIV-1 vectors has become available.24 In this cell line the production of the gag–pol and the
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Figure 1
Production of HIV-1-based lentiviral vectors. The figure
shows four constructs used to produce third generation lentiviral vectors. There are two packaging constructs, one expressing the gag and
pol genes and another expressing the rev gene. A third plasmid
expresses the VSV-G envelope protein. The vector plasmid contains
a chimeric LTR, consisting mostly of the Rous sarcoma virus (RSV)
LTR fused to the U5 region of the HIV LTR. The vector contains the ⌿
packaging signal that is absent in the packaging constructs (to prevent
packaging the RNA of the packaging constructs into vector particles),
and the rev responsive element, a promoter (P) and the transgene.
These four constructs are transiently transfected into 293-T cells and
the supernatant is harvested after approximately 48 h. The supernatant
can be concentrated 100 to 1000-fold by spinning in an ultracentrifuge. SD indicates splice donor, SA splice acceptor and CMV indicates
the promoter/enhancer from cytomegalovirus. Grey boxes in the vector indicate parts of the HIV-1 LTR. The 3′ LTR in the vector is of the
self-inactivating (SIN) variety and this LTR becomes the 5′ LTR as well
in the provirus, which integrates into the target cells. The figure is
modified from Dull et al.17
VSV-G proteins is controlled by the tet0 promoter, which is
regulatable, by doxycyclin. The gag–pol and the VSV-G plasmids are integrated into the genome of the packaging cell in
two separate places and their gene products can be induced
by doxycyclin treatment. Figure 2 shows a scheme demonstrating the generation of this cell line by Trono and colleagues. The advantages of producing vector particles from a
cell line with permanently integrated vector packaging
sequences is that reproducible high-quality, high-titer vector
supernatants can be made, and there is a lower theoretical
Leukemia
Figure 2
Production of a permanent packaging cell for HIV-1based vectors. In the first step the vector, packaging constructs and
the VSV-G envelope construct are transfected into 293 cells as
explained in Figure 1 to produce a cell line that can express the rev
protein and the envelope and gag–pol proteins upon induction. The
packaging cell line that is created in step 1 contains chromosome
integrated constructs that express the VSV-G and rev genes by a tetracyclin regulatable promoter and the tTA transactivator and gag–pol
genes are expressed by constitutive promoters. The gag–pol construct
is shown in Figure 1. The gag–pol transcripts are quickly degraded in
the nucleus without the rev post-transcriptional regulator. Therefore,
the gag–pol transcripts are stabilized upon rev induction. The gag–
pol and VSV-G genes have to be regulatable since their permanent
expression would lead to cell death. This cell line is now repeatedly
transduced by a vector from transiently transfected 293-T cells in
order to get multiple vector integration sites and as a consequence,
high-titer producers that can upon induction produce the viral proteins and the vector RNA (steps 2 to 3). In order to suppress expression
of the cytotoxic viral proteins the cells are grown in medium containing doxycycline while the cells are maintained. To produce vectors,
the viral proteins are induced in the absence of doxycycline. This
figure is modified from Klages et al24 where a description of the generation of the LVG packaging line was made.
risk of the undesirable recombination events that can lead to
the generation of replication-competent viral particles.
Lentiviral vectors can transduce quiescent hematopoietic
progenitor cells.25,26 However, it has also been demonstrated
that although human CD34+ cells in G0 can be transduced,
CD34+ cells in G1 or G2/S/M are transduced more efficiently.27
It has been shown that umbilical cord blood CD34+ cells that
are mostly in G0 and G1 can engraft efficiently in immunocompromised NOD/SCID mice, even after serial transplantation,14 and that lentiviral vectors can transduce these SCID
repopulating cells (SRCs) with 50–60% efficiency.18,19,21 The
umbilical cord blood (CB) cells that have been transduced are
mostly in G1 (65–70%) and to a lesser extent in G0 (20–
25%).14 It remains to be seen whether the high transduction
efficiency obtained with lentiviral vectors of these cells can
also be obtained when using the more therapeutically relevant
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N-B Woods et al
Lentiviral transduction of hematopoietic cells
Although lentiviral vectors do transduce non-dividing cells,
they unfortunately do not transduce all non-dividing cells. In
an experimental setting where human progenitor hematopoietic stem cells (PHSC) were transduced with lentiviral vectors
it was shown that: (1) lentiviral transduction is not inhibited
by aphidocolin (a cell cycle inhibitor); (2) transduced and
untransduced cells contained similar amounts of DNA per cell
reflecting that nondividing (N) and dividing (2N) cells were
similarly transduced; (3) transduction seemed to be independent of the S phase of the cell cycle,28 indicating that lentiviral vectors are able to transduce nondividing cells. The same
investigators noted, however, that transduction efficiencies
were markedly higher if the PHSCs had been cultured in cytokines for 48 h and also that the maximum percentage of cells
being transduced rarely exceeded 70%, even at high multiplicities of infection (MOI). In a follow-up study 1 year later,
they reported that lentiviral vectors preferentially transduce
cells in the G1 or S/G2/M phases of the cell cycle, while cells
in G0 were one-tenth as efficiently transduced.27 The authors
speculated that this depends on lower concentrations of
dNTPs in the target cells, which would limit the reverse transcription process. Subsequent studies in our laboratory in the
murine setting demonstrate that dNTPs added to the medium
only increase transduction efficiency by an extra 10%, suggesting other factors are preventing total transduction of primitive hematopoietic cells.29 This has implications for a gene
therapy setting in the hematopoietic system, firstly because of
the sources of stem cells available. Hematopoietic cells can
be derived from cord blood (CB), bone marrow (BM) or mobilized peripheral blood (PB). Most of the work with lentiviral
transduction of hematopoietic cells has been performed using
CB, but autologous BM cells or PB cells are more likely to be
used in human gene therapy trials. However, both BM and
PB have fewer cells in G1 and more in G0 than CB does. It
might be necessary to adapt the transduction protocol to these
sources by optimizing culture conditions, such as cytokine
stimulation or culture time. Second, it will be critical not to
stimulate the cells too much, since even stimulation from G0
to G1 curtails the engraftability of these stem cells.7,8,15 It may
be argued that the main limitation for successful gene therapy
of blood disorders lies in the vector’s efficiency to transduce
hematopoietic stem cells. Lentiviral vectors do offer distinct
advantages over oncoretrovirally based vectors, but the full
potential of gene therapy will only be met with further modifications to the vectors rendering them truly capable of
efficient stem cell transduction.
The key characteristic of the lentiviral vector which distinguishes it from the standard vectors based on oncoretroviruses
is that it allows for the infection of primitive nondividing stem
cells. The mechanisms behind this central feature, which is to
transport the proviral DNA through an intact nuclear membrane, for subsequent integration into the host DNA, remains
to be elucidated. During lentiviral infection, both the genome
and a number of viral proteins are actively transported into
the nucleus via a nuclear pore. It is hypothesized that the proteins and the vector genome form a preintegration complex
which contains a number of amino acid sequences which
localize the complex to nucleus known as the nuclear localization signal (NLS). In 1993 the lentiviral gag matrix protein
was implicated in this process.30 The investigators managed
to isolate a sequence of the gag matrix protein that would
direct nuclear import when conjugated to a heterologous protein. They also showed that a mutant HIV-1, carrying an
amino acid substitution in this NLS, would not replicate in
growth-arrested cells. Later, another group showed that the
viral protein R (vpr) is also important in the viral nuclear
import process.31 The efficiency of the nuclear localization
might be another factor influencing the transduction efficiency
seen by lentiviral vectors. The lentiviral vector is in large
stripped of its original wt genes, but in an attempt to increase
the transduction efficiencies, efforts have been made to identify the NLS in the wt HIV-1 genome and reinsert those to the
vector. While the roles of both vpr and the gag matrix protein
as NLSs remain controversial,32,33 a sequence within the pol
gene known as the cPPT-CTS fragment or the DNA flap, has
been shown to be a cis-determinant of HIV-1 DNA nuclear
import.34 When the DNA flap was inserted into a lentiviral
vector, transduction efficiencies with the new vector were significantly higher compared to a vector without the DNA flap.
For example, 45% of primary human lymphocytes could be
transduced with the DNA flap-containing vector, while the
original vector only transduced 15% of the cells. When
targeting CD34+ CB cells, the DNA flap-containing vector
transduced two- to three-fold more cells than did the
control vector.35,36
Lentiviral vector transgene expression
While optimizations of the lentiviral vector and lentiviral
transduction conditions have allowed for improved transduction of SCID repopulating cells, advances were simultaneously
being achieved with expression of the transgenes. Lentiviral
vectors as they were first developed for gene therapy applications have transgene expression driven from ubiquitously
expressing internal promoters such as the cytomegalovirus
(CMV) early promoter, or the murine phosphoglycerate kinase
(PGK) gene promoter.18,19 While these constructs were hailed
a success, as they showed persistent expression in serially
transplanted NOD/SCID mice, the level of transgene
expression was low. In contrast to the HIV-based vectors driving expression from the wt HIV LTR, which have been shown
to be capable of high-level transgene expression,37,38 safety
considerations will likely prevent their use in clinical settings.
The HIV-1 LTR promoter which requires the expression of the
hematologically and neurologically toxic tat protein trans-activator for expression would require the vector to contain the
tat gene along with the therapeutic gene.39,40 Additionally, the
LTR promoter and tat gene contribute a nonessential extra
500 bp of DNA to the vector with sequence homology to the
wt HIV and therefore carries an increased risk of homologous
recombination with a patient who already has or acquires wt
HIV-1 infection. However, in hematological disorders where
high-level transgene expression is required in many cell types
or in diseases where transgene expression is necessary at an
early progenitor cell level, efficient ubiquitously expressing
promoters are essential. These ubiquitously expressing promoters must function within the lentiviral vector to produce
long-term expression in vivo throughout hematopoietic differentiation into mature hematopoietic lineages. A number of
candidate promoters have emerged, for example, the hybrid
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bone marrow (BM) and peripheral blood (PB) CD34+ cells,
since these latter cell populations have a much higher proportion of G0 cells than their CB counterparts.7,8,14 We will
now review the available data and describe what challenges
lie ahead.
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Leukemia
chicken actin promoter containing the CMV enhancer region
(CAG) and the human elongation factor-1␣ (EF-1␣) gene promoter.41 Oncoretroviral LTR promoters from viruses such as
the murine stem cell virus (MSCV) or gibon ape leukemia virus
(GALV), have also been tested with regards to expression in
hematopoietic progenitors.42 The MND promoter from a
modified myeloproliferative sarcoma virus (MPSV) LTR43 has
also been tested within a bovine immunodeficiency lentiviral
vector.44 Testing of these vectors in vitro on human hematopoietic progenitor cells led to the conclusion that high-level
transgene expression from a lentiviral vector could be achieved and that most probably the highest expressing vector was
that driving expression from the EF-1␣ promoter.41,42 Subsequent analyses of expression in vivo in the progeny cells
from transduced SRC reveal that the EF-1␣-containing vectors
yield high-level expression of the hematopoietic lineages with
levels seven- to eight-fold higher than the original PGK lentiviral vector.45 Studies using murine lin−, c-kit+ sca+ progenitor
cells show long-term high-level expression without loss of
expression in all hematopoietic lineages even after serial
transplantation.46
A lentiviral vector which generates extremely high
expression levels of the erythroid specific ␤-globin transgene
has recently been demonstrated in mice where the level of
expression is similar to that found in normal erythroid cells.20
The vector uses the human ␤-globin promoter and a truncated
version of the ␤-globin locus control region to drive
expression of the ␤-globin gene, generating high expression
levels that are tissue-specific and restricted to the erythroid
lineage.
Further modifications to the lentiviral vector were also performed in conjunction with the promoter in order to increase
expression levels. Such modifications were the addition of the
woodchuck post-transcriptional regulatory element (WPRE),47
and the removal of the U3 element in the HIV-1 LTR of the
vector rendering the vector self-inactivating (SIN vector).48
The WPRE DNA sequence when placed immediately downstream of the transgene in the vector is thought to increase
the stability of the mRNA after transcription. It may also play
a role in increasing the efficiency of mRNA processing. Using
the WPRE within the lentiviral vector resulted in variable
increases in transgene expression in in vitro studies on human
hematopoietic cells.41 The degree of expression seemed
dependent on the regulatory elements nearby in the lentiviral
vector. For example, expression levels from vectors containing
the weaker promoters, PGK and CMV, could be dramatically
increased with the WPRE, whereas expression levels were
marginally improved or even slightly reduced in vectors containing stronger promoters like CAG and EF-1␣. Other studies
using oncoretroviral vectors also show the context dependence of expression using the WPRE.49 The SIN vectors were
originally designed to increase the biosafety of lentiviral vectors by preventing full-length transcription of the proviral vector genome. This reduces the chances of homologous recombination with the wild-type HIV. However, the SIN deletion
in which 8 Sp1 transcription factor binding sites are removed
may hypothetically influence the level of transgene expression
from the vector, particularily in situations where the internal
promoter also contains multiple Sp1 sites.50,51 Interestingly,
the expression levels generated by SIN vectors were also
dependent on other regulatory elements present in the vector.
For example, in primary human CD34+ cells transduced with
the PGK and the PGK SIN vectors, the SIN vector generated
only half of the expression level compared with the standard
PGK vector. However, in the context of the EF-1␣, the SIN
vector increased the expression 1.5-fold.41
Although thorough analyses of expression from several vectors were performed in vitro, follow-up experiments would be
necessary to confirm the ubiquitous expression in vivo
throughout hematopoietic differentiation. Experiments in the
NOD/SCID mouse demonstrated a high degree of correlation
between the in vitro and in vivo expression results where
high-level transgene expression could be obtained in all hematopoietic lineages.45 The comparability of the results also
suggests that the rapid screening of new therapeutic vectors
driving expression from ubiquitously expressing promoters
should be possible in vitro prior to testing in animal models.
One feature of the EF-1␣ promoter is that it contains a
942 bp intron where multiple transcription binding sites are
located. Occasionally, during vector production in packaging
cell lines, the intron is spliced out of the vector RNA prior to
packaging.42 The spliced vector results in transgene
expression being driven from a truncated promoter and studies have shown that the spliced EF-1␣ promoter does not
express as well as the non-spliced variant. However, persistent
moderate expression can be achieved long term in vivo in
the progeny of NODSCID repopulating cells using a truncated
(200 bp) EF-1␣ promoter. Therefore, expression from the EF1␣ promoter may further be optimized by the mutation of the
splicing donor and acceptor in the EF-1␣ promoter, thus
preventing its truncation during vector production in the
packaging cell line.
Biosafety
The third generation lentiviral vector packaging system shown
in Figure 1 contains only three of the nine genes in HIV-1,
gag, pol and rev. Sixty percent of the viral genome has been
removed in the packaging system and this completely
excludes the possibility of generating wild-type HIV-1. Since
the packaging system shown in Figure 1 has four separate
plasmids, the probability of producing a replication-competent recombinant (RCR) is very low. Self-inactivating (SIN)
vector systems minimize the likelihood of generating RCRs
even further. However, it will be difficult to produce SIN vectors with high titer by transfecting a SIN vector plasmid (intact
5′LTR and 3′ SIN LTR) into the stable packaging cell line as
described in Figure 2, because the plasmid will most likely
not integrate into chromosomal sites that are favorable for
high-level expression. High titers can only be produced by
transducing the packaging cell line using a vector genome
with a promoter that can generate the full genomic vector
RNA. The lentiviral vector packaging cell line can be used to
produce lentiviral vectors that should theoretically be as safe
or safer than current oncoretroviral vectors. However, vectors
produced in stable producer cell lines generated in the future
will have to be tested for the presence of RCR and toxic products before such lines can be used to produce vectors for the
clinic. Lentiviral vectors other than those based on HIV-1 have
been made although their development is less advanced at
present. These are HIV-2 vectors and vectors based on the
feline immunodeficiency virus (FIV), bovine immunodeficiency virus, simian immunodeficiency virus (SIV) and the
equine infectious anemia virus (EIAV).44,52–55 It has been suggested that these alternative lentiviral vectors may be safer
because they are less pathogenic in humans (HIV-2) or are
not known to cause human disease. However, it is likely that
the greatest risk of using lentiviral vectors in the future is going
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N-B Woods et al
Future challenges
While it has been shown that lentiviral gene transfer into HSCs
from umbilical cord blood has been demonstrated to be
efficient when tested in the NOD/SCID mouse, less is known
about the gene transfer efficiency into BM and PB-derived
HSCs. While it is likely that efficient gene transfer protocols
into these cells can be developed, the transduction conditions
must be defined more accurately. Similarly, the transduced
HSCs will have to be tested in an autologous transplantation
setting in both humans and large outbred animals to test
whether long-term reconstituting HSCs can be effectively
transduced. So far, the vectors that have been tested in HSCs
are mostly single gene vectors. For effective testing of therapeutic vectors it will be important to have lentiviral vectors
containing two transcriptional units that can express the therapeutic gene and a selectable marker gene to sufficiently high
levels using a safe packaging system. Figure 3 shows a vector
that uses an active promoter in the middle of the HIV-1 LTR
(the promoter replaces the U3 portion in the LTR) to drive the
selectable gene and an internal promoter to drive the therapeutic gene. This vector can be produced in producer cells
according to the principles described in Figure 2. Vectors
where promoters are used to replace the U3 region in HIV-1
have been made using the cytomegalovirus and the murine
embryonic stem cell virus promoters/enhancers and have
been shown to work well in human cells.24,42 While the concepts have been developed for the safe production of lentiviral
vectors, the new permanent packaging cells will have to be
Figure 3
Examples of vectors (shown as integrated proviruses)
expressing two genes, for example, a therapeutic gene and a selectable marker gene. (a) A self-inactivating (SIN) vector where most
of the U3 region of the LTR has been deleted. An internal promoter
(P) drives the expression of two genes, 1 and 2. The advantage with
this design is that the SIN design offers increased safety but the disadvantage is that the expression of gene 2 is going to be reduced since
it is placed downstream of an internal ribosomal entry site (IRES)
which allows the translation of the second gene from the bicistronic
mRNA. The SIN vector cannot be produced in the permanent packaging cell line shown in Figure 2. (b) This vector contains two promoters,
one in the LTR (not the HIV-1 promoter for safety reasons) and another
promoter (P2) internally. The advantage with this design is that the
expression level will be as high as expected from the two individual
promoters and here the vector can be produced in the permanent
packaging cell line shown in Figure 2. Since the LTR contains an
active promoter there is increased risk from insertional mutagenesis
compaired with the construct in (a).
tested to assess the quality and safety of the vector preparations that will be generated using these lines. Most
importantly, suitable candidate diseases will have to be selected to test the efficiency of lentiviral vector gene therapy. To
begin with, it would be wise to select a disease where correction of a fraction of the patients cells would be sufficient to
generate a therapeutic effect and where relatively low levels of
the transgene product will be sufficient to correct the disease
phenotype. Thereafter, more ambitious goals can be pursued
where high level expression of the therapeutic gene is
required or where a very large fraction of the patient’s stem
cells need to be corrected, requiring either a very high transduction efficiency or an effective selection of the transduced
cells, for example, by a vector gene product that generates a
selective advantage in vivo.56
Recently, several studies have been published which demonstrate stem cell plasticity. Hematopoietic stem cells have
been shown to be able to generate heart muscle following
myocardial infarction, and liver damage has been repaired in
a similar experiment where hematopoietic stem cells were
transplanted to mice with liver disease.57,58 This stem cell
plasticity has implication for the development of cell and gene
therapy since it may be possible in the future to generate several separate tissues from one type of adult stem cell which
was previously considered to be specialized to the generation
of one tissue. An alternate approach would be to deliver genes
to more than one type of stem cell to cure a disease, for
example, hematopoietic stem cells and mesenchymal stem
cells to correct some lysosomal storage disorders where there
are defects in hematopoietic cells and connective tissue. In
the latter case, lentiviral vectors have an advantage as a gene
delivery vehicle to various types of stem cells since some tend
to be quiescent and others rapidly dividing. While oncoretroviral vectors can only transduce dividing cells, lentiviral vectors can transduce both dividing and quiescent cells. Therefore, lentiviral vectors are versatile gene delivery vehicles and
can be used to transfer genes to many types of stem cells.
Rapidly dividing ES cells are efficiently transduced by lentiviral vectors and reduction in lentiviral transgene expression following differentiation of ES cells is considerably less pronounced than the complete silencing or shut-off seen from
oncoretroviral transgenes.59 ES cells may be used in the future
to generate specific tissues for cell and gene therapy, and here,
lentiviral vectors may be useful for sustained transgene
expression.
To conclude, lentiviral vectors are very promising tools for
the development of gene therapy using hematopoietic or other
types of stem cells as targets. To date, it seems that lentiviral
transgenes can be expressed at high levels over time, both
when strong general promoters and tissue-specific control
elements are used. Therefore, lentiviral vectors will probably
be used in many gene therapy approaches in the future within
and outside the hematopoietic system.
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SPOTLIGHT
to be insertional mutagenesis, the same main risk as is likely
to ensue with the use of oncoretroviral vectors. Therefore, it
is likely that the HIV-1-based vectors will have similar biosafety risks as the other lentiviral vectors mentioned. This discussion presumes that effective testing for microorganisms,
toxic products and RCRs can be developed for the lentiviral
producer cells as has been possible for the oncoretroviral
producers.
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