ARTICLE
Auteur(s) : Gaetano O1 Pier Paolo
CLAUDIO2 Tiziana TONINI2 Antonio
GIORDANO2
1 Department of Neurosurgery, The Farber
Institute for the Neurosciences, Jefferson Medical College, Thomas
Jefferson University, College Building, Suite 501, 1025 Walnut
Street, Philadelphia, PA 19107, U.S.A. 2 Sbarro
Institute for Cancer Research and Molecular Medicine, College of
Science and Technology, Department of Biotechnology, Temple
University, Philadelphia, PA 19122, U.S.A.
Reprints: G. o Fax: (+1) 215‐9552992 E‐mail:
Gaetano.ojefferson.edu
Article accepted on 11\6\2003
Key words: The lentiviral vector system based on the
human immunodeficiency virus type 1 (HIV‐1) has attracted much
attention in the field of gene therapy [1‐3]. The latest generation
of HIV‐based vectors is an efficient tool for the in vitro
transduction of many cell types, including non‐dividing cells and,
more importantly, various types of stem cells [3‐11]. Many studies
are currently ongoing to apply lentiviral‐mediated gene transfer in
preclinical models for neurodegenerative diseases [4, 12‐15],
genetic disorders [16‐21], cancer [22‐24], diseases of the kidney
[25], acquired immunodeficiency syndrome (AIDS) [26] and
cardiovascular illnesses [27]. Indeed, HIV‐based vectors have been
used with success for in vivo gene delivery in animal models
[3, 4, 12‐15, 19‐21, 25, 27]. In these studies, lentiviral vectors
were infused into different organs, such as brain [4, 12‐15], lungs
[19], vascular system [21, 27], kidneys [25] and subcutaneous tumor
deposits in nude mice [23]. Interestingly, a recent preclinical
study has applied lentiviral‐mediated gene transfer to restore the
expression of type VII collagen into keratinocytes and fibroblasts,
which derived from patients with dystrophic epidermolysis bullosa
[18]. This pathological condition is caused by an inherited genetic
disorder, which is responsible for the depletion of type VII
collagen. The lack of expression of type VII collagen results in
the detachment between epidermis and dermis. Sadly, there is no
cure for this disorder. The expression of lentiviral‐encoded type
VII collagen has corrected the phenotype of keratinocytes and
fibroblasts, in terms of morphology, proliferative potential,
motility and attachment to matrix [18]. In addition, genetically
modified cells were implanted into immune deficient mice. The
engrafted human cells could generate in vivo anchoring
fibrils at the dermal‐epidermal junction [18]. This study has
indeed provided an encouraging result for the potential treatment
of dystrophic epidermolysis bullosa. In another study, HIV‐derived
vectors encoding for human erythropoietin (EPO) were injected into
human skin grafted on immune deficient mice [28]. Following a
single administration of lentiviral vectors, the grafted human skin
released EPO to the bloodstream of mice, indicating that skin can
allow for expression of lentiviral‐encoded therapeutic factors
[28]. Although preclinical studies seem very promising, the
possible employment of HIV‐based vectors in clinical trials is a
very controversial issue [29, 30]. In the year 2001, the
Recombinant DNA Advisory Committee of the National Institutes of
Health rejected the first HIV‐based gene therapy protocol for the
treatment of AIDS [29]. There were many concerns in the matter of
safety. These concerns were in part justified by the lack of
clinical experience to predict possible adverse effects induced by
gene transfer [2]. For instance, the development of a leukemia‐like
illness in two of the eleven children of the gene‐based trial of
severe combined immunodeficiency (SCID)‐X1 has recently caused a
setback for gene therapy clinical trials [31‐36]. The blood
neoplasia derived from the random insertion of the retroviral
transfer vector into the LMO‐2 locus of the short arm of
chromosome 11 [37]. The over‐expression of LMO‐2 was
observed in acute T cell lymphoblastic leukemia, following the
chromosomal translocation t(11;14) [39]. Indeed, the risk for
insertional mutagenesis was known [2, 39], however, the extent of
such an incidence was unpredictable. Despite the strides achieved
in the field of cancer research, we still do not have the tools for
an effective preventive cancer prognosis [2]. On these grounds, it
is not possible to assess properly the risk to benefit ratio for
all patients in gene therapy interventions. In addition to these
safety issues, HIV‐based vectors pose many other concerns, as they
derive from a pathogenic agent that is lethal in humans. Also in
these cases, the risk assessment is very difficult, as many aspects
of AIDS pathogenesis have not been completely elucidated. This
review will mainly focus on safety concerns associated with the
hypothetical use of HIV‐based vectors in humans. The properties and
drawbacks of the currently available viral and non‐viral vector
systems have been described in Table
I.Table I. Description of the principal
gene delivery systems
| Vector system |
Characteristics |
Drawbacks and possible adverse effects in therapeutic
applications |
| Retroviral vectors |
They require the breakdown of the nuclear membrane to access
the cellular chromosomal DNA, therefore, they can only transduce
dividing cells. They allow for long‐term transgene expression due
to the integration of the retroviral vector into the cell genome.
Retroviral vector particles can carry up to 9 kb of chimerical
viral genome. They can be pseudotyped with amphotropic retroviral
or VSV‐G envelopes: this confers a broad cell tropism. They can be
produced with relatively high titers
(106‐107 tu\ml), and can be easily
purified via high speed centrifugation if pseudotyped with VSV‐G
envelopes. Both murine and avian retroviruses are distantly related
to primate retroviruses: this contrives in minimizing interactions
with HERVs. |
Not suitable for the transduction of non‐dividing cells.
Homologous recombination events may generate replication competent
retroviruses. The integration of the retroviral vector occurs
randomly in the cell genome: this may cause insertional mutagenesis
events. Complement‐mediated lysis and other humoral immune
responses destroy rapidly retroviral vector particles in
vivo. |
| Lentiviral vectors based on HIV‐1 |
They can also transduce non‐dividing cells. Long‐term transgene
expression due to the integration of lentiviral vector into the
target cell genome. FLAP or cPPT HIV‐1‐derived vectors are one of
the best developed viral vector systems: they allow for high
transduction efficiency, long term and high levels of transgene
expression. HIV‐1‐derived vectors can carry up to 10 kb of
chimerical viral genome. They can be pseudotyped with amphotropic
retroviral or VSV‐G envelopes: this confers a broad cell tropism.
They can be produced with relatively high titers
(106‐107 tu\ml), and can be easily
purified via high‐speed centrifugation if pseudotyped with VSV‐G
envelopes. |
Subjects are seroconverted to HIV‐1. Possible insertional
mutagenesis in target cells. The close relation of HIV‐1 to
human retroviruses increases the possibility of interactions with
HERVs. Packaging cells express HIV‐1 tat and rev regulatory
proteins. Homologous recombination events may generate replication
competent lentiviruses. Complement‐mediated lysis and other humoral
immune responses destroy rapidly lentiviral vector particles in
vivo, after being pseudotyped with amphotropic retroviral or VSV‐G
envelopes. |
| Lentiviral vectors based on FIV, EIAV and BIV. |
They can also transduce non‐dividing cells. They are not
pathogenic in humans, therefore the seroconversion of the subject
to these lentiviral vectors does not pose an issue. These
lentiviral vector systems are distantly related to primate
retroviruses: this contrives in minimizing interactions with HERVs.
They can be pseudotyped with amphotropic retroviral or VSV‐G
envelopes: this confers a broad cell tropism. Long‐term transgene
expression due to the integration of lentiviral vector into the
target cell genome. They can be produced with relatively high
titers (106‐107 tu\ml), and can be
easily purified via high‐speed centrifugation if pseudotyped with
VSV‐G envelopes. FIV‐, EIAV‐ and BIV‐based vectors can take up to
10kb of chimerical viral genome. |
Possible insertional mutagenesis in target cells. Lentiviral
regulatory proteins are present in packaging cells. Homologous
recombination events may generate replication competent
lentiviruses. Transduction efficiency, levels of transgene
expression and duration of transgene expression are still rather
sub‐optimal, if compared to the latest generations of HIV‐1‐derived
vector systems. |
| Adeno‐associated viral (AAV) vectors |
They can transduce cells at any cell cycle phase. They have a
rather broad cell tropism. They can be produced at high titers
(1010 tu\ml). The integration of viral genome
allows for stable transgene expression. They are non‐pathogenic and
non‐toxic. The generation of AAV vector stocks no longer requires
helper viruses. AAV‐based vectors can be easily and efficiently.
Purified with columns, avoiding CsCl purification system. |
There is a limited capacity for foreign genes (about
4 kB), as they have a small genome (5kb). Although wild type
AAV has a specific and safe site of integration in the cellular
chromosomal DNA, recombinant AAV‐based vectors integrate their
genome randomly into the DNA of target cells, generating the
possibility of insertional mutagenesis. Humoral host immune
responses neutralize AAV‐based vectors in vivo. |
| Adenoviral vectors |
They can transduce cells at any cell cycle phase. Both
transduction efficiency and levels of transgene expression are
high, but only transient. Suitable for genetic immunization
programs. They can be easily produced at very high titers
(1012 tu\ml). They have a rather broad cell
tropism. Adenoviral vector particles can accommodate large
transgenes, provided proper deletion of adenoviral vector genome
(up to 7 or 8kb of foreign DNA sequence can be just added to
the vector). |
Cytotoxic T lymphocyte (CTL) and humoral immune responses may
cause serious adverse effects in patients and the depletion of
transduced cells. Host humoral immune responses destroy rapidly
adenoviral vector particles in vivo. Not suitable for
long‐term transgene expression. |
| Non‐viral vector systems: cationic liposomes or DNA‐protein
complexes |
They are not based on infectious agents. They can carry large
DNA sequences. They are suitable to deliver oligonucleotides. They
allow for the transfection of a wide variety of cell types.
Suitable for genetic immunization programs. A wide variety of cell
types can be transfected. |
They lack specific cell targeting. Transfection efficiency
might not be very high. Transgene expression is only transient. In
vivo applications can be problematic. Unmethylated CpG sequences of
bacterial plasmid DNA elicit host immune responses. They are not
suitable for long‐term transgene expression. |
(Abbreviations: tu\ml ∓ transducing units per milliliter;
VSV‐G ∓ vesicular stomatitis virus G protein;
HERVs ∓ human endogenous retroviruses;
FIV ∓ feline immunodeficiency virus;
EIAV ∓ equine infectious anemia virus;
BIV ∓ bovine immunodeficiency virus). .
The engineering of the latest generation of HIV‐based
vectors
As already mentioned, the latest generation of HIV‐based vectors
can transduce very efficiently many cell types [1‐11], and works
very well in animal models [4, 12‐15, 19‐21, 25, 27]. These
HIV‐based vectors have been termed either as cPPT or FLAP vectors.
The cPPT or central DNA FLAP sequence is composed of a 118‐bp
polypurine tract followed by a central termination sequence, which
is in the HIV‐1 pol gene [40, 41]. Intriguingly, this
simple and short DNA sequence mediates the nuclear import of the
HIV‐1 preintegration complex [40, 41]. The insertion of the
central DNA flap has drastically enhanced the transduction efficacy
of the latest generation of HIV‐based vectors. Interestingly, a
central DNA flap sequence is present in all lentiviruses [40]. A
typical FLAP or cPPT HIV‐based vector is shown in figure 1. In this transfer
vector, the central FLAP sequence is located downstream of the
packaging signal (Ψ) and the rev response element (RRE). An
internal promoter drives the expression of the transgene, which can
be accommodated into the polylinker sequence that is downstream of
the internal promoter. Usually, HIV‐based vectors contain a CMV
promoter for enhanced transcriptional activity [2, 6]. However,
many types of internal promoters can be used, including inducible
or tissue specific promoters [2, 36]. The latest generation of
HIV‐based vectors is based on a self‐inactivating form of proviral
vectors [2, 6]. This means that after the viral‐mediated gene
delivery, the 5‘ long terminal repeat (LTR) does not transcribe
[42]. This can be achieved by deleting the promoter\enhancer region
from the 3‘ LTR (Fig.
1) [42]. The 5‘ LTR is transcriptionally active in the
proviral form. Following the packaging of the viral mRNA into the
virion and the infection of the target cell, the viral mRNA is
reverse transcribed in the cell cytoplasm. At this point in time,
the reverse transcription process generates a double stranded viral
cDNA in which the 5‘ LTR is the exact copy of the 3‘ LTR [42]. For
this reason, the 5‘ LTR no longer transcribe in transduced cells
[2, 42]. Self‐inactivating lentiviral and retroviral vectors are
particularly suitable to deliver transgenes under the control of
tissue specific or inducible promoters, as the inactivation of the
5‘ LTR eliminates transcriptional interference between the
promoters [2]. In addition, self‐inactivating versions of
retroviral and lentiviral vectors may minimize the risk of
insertional mutagenesis, as the 3‘ LTR is not transcribing [42].
This should prevent the expression of cellular oncogenes that may
be present downstream of the integration site of the transfer
vector [2, 36].
.
The latest generation of HIV‐based vectors seems to be the most
suitable tool to manipulate various types of stem cells [1‐11]. The
presence of the cPPT or central DNA flap allows for efficient gene
transduction of many cell types. The levels of transgene expression
are enhanced by the transcriptional regulatory element of woodchuck
hepatitis virus (WPRE) [2, 6, 36], which is located downstream of
the transgene (Fig.
1). The latest generation of HIV‐based vectors has also
shown a stable duration of transgene expression [43, 44].
Safety issues for HIV‐based vectors
The assessment of the risk to benefit ratio is the central issue
for human gene therapy protocols [2]. As already mentioned, the
lack of clinical experience does not allow for proper evaluation of
the risks involved in gene‐based interventions. Under these
conditions, the highest risk should always be assumed, in order to
minimize the possible harm that patients may sustain in the
procedure. Relatively healthier patients are therefore excluded
from gene therapy clinical trials. These criteria are applied to
gene delivery systems that are based either on non‐pathogenic or
minimally pathogenic viruses. Therefore, both ethical and practical
issues oppose the application of HIV‐based vectors in clinical
trials. It has been argued that a gene delivery system cannot be
based on an infectious agent that is pathogenic for humans,
especially if the pathogenesis of the disease is still not
completely clear and is not curable. Under these circumstances, the
assessment of the risk to benefit ratio is almost impossible.
The first concern in the matter of HIV‐based vectors is the
seroconversion of the subject to certain components of HIV‐1. In
addition, if the vector will be administered in patients with AIDS,
the risk of a recombination event between the vector and
HIV‐1 should be considered. The resulting infectious agent
might even be more pathogenic than HIV‐1 itself. At this
stage, it is not possible to assess the entity of such a risk
[29].
The production and manipulation of HIV‐based vectors must be
carried out either in biosafety laboratory level three (BL3)
facilities, or in BL2 facilities using BL3 precautions. These
manipulations are very cumbersome for producing large quantities of
clinical‐grade lentiviral vector stocks. In addition, scaling up
the production of HIV‐based vectors may increase the possibility of
generating replication‐competent viruses by homologous
recombination. The presence of a viral envelope with broad cell
tropism will generate a novel infectious agent with a completely
unpredictable pathogenicity in the host.
Other lentiviral vector systems are emerging, which are based on
lentiviruses that are not pathogenic in humans, such as feline
immunodeficiency virus (FIV) [45, 46], equine infectious anemia
virus (EIAV) [44], bovine immunodeficiency virus (BIV) [47] and
sheep Visna virus [43]. The eventual employment of these lentiviral
vectors in gene therapy clinical trials would circumvent the
critical issue of HIV‐seroconversion of the subject. Moreover,
FIV‐, EIAV‐, BIV‐ and Visna viral‐based vectors can be handled in
BL2 facilities. This greatly facilitates the production of large
quantities of clinical‐grade lentiviral vector stocks. FIV‐, EIAV‐
and BIV‐based vectors have been used with success in a variety of
preclinical studies [46, 48‐52]. FIV‐mediated in vivo gene
transfer corrected a cystic fibrosis defect in a rabbit animal
model [46, 48], a lysosomal storage disease called Sly syndrome in
mice [49], and could efficiently transduce a variety of retinal
cells in a nonhuman primate model [50]. EIAV‐based vectors
pseudotyped with rabies virus glycoprotein were injected into the
striatum of rats allowing for an efficient transduction both at the
site of injection and of distal neurons [51]. BIV‐based vectors
could efficiently transduce ocular cells in a murine animal model
[52]. Taken together, these findings seem to indicate that FIV‐,
EIAV‐ and BIV‐mediated gene transfer may be an alternative to
HIV‐derived vectors.
Another interesting aspect is that FIV, EIAV, BIV and Visna virus
are distantly related to primate lentiviruses [43]. This minimizes
the possibility to have recombination or other types of
interactions between the vector and human endogenous retroviruses
(HERVs) [36]. This is obviously in contrast to HIV‐based vectors.
It was reported that HIV‐1 and the HERV‐K family might even
share a common phylogenetic ancestry [53]. This group of HERVs
entered the human genome approximately 30 million years ago
[53]. It was observed that more than 70% of patients with AIDS test
positive for antibodies to HERV‐K structural proteins [54]. These
antibodies are normally absent or eventually expressed at very low
levels in the general population [54]. In vitro studies also
reported that HIV‐1 Rev protein recognizes the
sequence‐specific nuclear RNA export factor of the HERV‐K family
[53, 55]. These findings indicate that HIV‐1 infection
promotes the expression of HERV‐K structural proteins in humans.
Studies are currently ongoing to determine whether the expression
of HERV‐K proteins may in part contribute to the progression of
AIDS [53]. The pathological potential of HERVs cannot be predicted,
especially if they should recombinate with HIV‐1 sequences to
give rise to a replication‐competent retrovirus. The biology of
HERVs is indeed very complex and deserves further investigation
[56]. HERVs sequences constitute at least 1% of the human genome
and are implicated in a variety of biological processes, such as
protection of cells from superinfection by exogenous retroviruses,
protection of the embryo from retroviral infection (germ line
vaccination) and from maternal immune responses, cell fusion,
tissue‐specific gene expression, alternative splicing and
polyadenilation. On the other hand, abnormal expression of HERVs
protein was detected in malignancies and autoimmunodiseases [56].
Studies are currently in progress to establish a possible
involvement of HERVs in these pathological conditions [56].
Most of HIV‐1‐based vectors are Rev‐dependent at the packaging
stage. Rev has two functions. First, it promotes the nuclear export
of lentiviral RNA, by binding to the Rev response element (RRE)
[57]. The second function of Rev consists of stabilizing lentiviral
transcripts [58‐60]. The codon usage of HIV‐1 and of simian
immunodeficiency virus (SIV) has a different bias from that of
human genes [59, 60]. This is due to the fact that the genomes of
HIV‐1 and SIV are AT rich. The presence of Rev and RRE allows
for a more efficient gene expression of lentiviral components,
especially gag, pol and env. HIV‐based vectors
are produced transiently, by co‐transfecting four plasmids into a
highly transfectable cell line [2, 61]. These plasmids encode for
HIV‐1 gag‐pol, HIV‐1 Rev, the vesicular stomatitis
viral G (VSV‐G) envelope and the transfer vector carrying the
transgene of interest. Only the transfer vector contains the
packaging signal, therefore, Rev protein is only expressed at the
packaging stage. After the formation of the recombinant virion, Rev
protein is no longer present [2, 61]. However, there is still the
risk of a passive transmission of the plasmid encoding for Rev
protein into the subject. For instance, a study reported the
presence of the retroviral 4070A envelope DNA in the vector
supernatant [62]. This finding indicates that the plasmid DNA used
in the transient co‐transfection of the packaging cell line is not
completely removed from the viral supernatant. Another possible
route of transmission of Rev into the subject may derive from
homologous recombination events between the Rev and packaging
constructs. Such a possibility should be remote, as the various
constructs have been engineered to minimize overlapping sequences.
In addition, the transient nature of the production of the
lentiviral vector stocks greatly minimizes the possibility of
homologous recombination events [2, 61]. Some studies have achieved
the engineering of rev‐independent HIV‐based vectors, by optimizing
the codon usage according to the human system [59, 60]. However,
these studies were not conducted on the cPPT or FLAP HIV‐based
vectors. Therefore, the transduction efficiency was not
optimal.
Another critical aspect of in vivo gene delivery is the
possible transmission of exogenous DNA to the germ line. This was
observed in a preclinical study involving adenoviral vectors [63].
The majority of the mice used in this study tested positive for
adenoviral DNA in the ovaries or testis. After mating these
animals, there was no evidence of transmission of adenoviral DNA in
the offspring [63]. Most likely, this finding is due to the lack of
integration of adenoviral genome into the cellular chromosomal DNA
[2]. Viral vectors that can integrate their genome into the host
chromosomal DNA have higher probability of entering the germ line.
This is indeed one of the major drawbacks for in vivo gene
delivery of integrating viral vectors. Every precaution must be
taken to avoid the transmission of HIV‐related sequences to the
germ line, as they may interfere with the biological functions of
HERVs.
Conclusion
The HIV‐based vector system is by far the best developed among
the various lentiviral vectors [43]. However, a variety of safety
and ethical concerns preclude the employment of HIV‐based vectors
in the clinical setting. Safety became the most pressing issue for
gene therapists after the adverse reaction that occurred in two
children of the gene‐based SCID trial. The field of vector design
is currently improving vector systems that are based on
lentiviruses that are not pathogenic in humans, and that are
distantly related to primate retroviridae. Probably, these
types of lentiviral vector systems will be safer than HIV‐based
vectors. In addition, the application of self‐inactivating
retroviral and lentiviral vectors may also reduce the risk of
insertional mutagenesis [36]. Indeed, the engineering of safer and
more efficient lentiviral vectors will be very useful to implement
cell transplantation therapy and the genetic engineering of stem
cells of various derivations. The optimization of stem cell
transplantation techniques may eventually lead to more effective
therapeutic approaches for the treatment of neurodegenerative
disorders, autoimmune diseases and immunodeficiencies.
Acknowledgements. The authors wish to thank Marie Basso for
helpful comments on the manuscript. This work was supported by NIH
grants, by "The Farber Institute for the Neurosciences" and by the
"Sbarro Health Research Organization".
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