INTRODUCTION
Gene therapy is a promising novel approach to the treatment of a variety of disorders including infectious, genetic and neurologic diseases and cancer (1) (2) (3) (4) (5) . It employs various methods to deliver foreign genes into somatic cells with the ultimate goal of incorporation and stable expression of the gene of interest. Methods used for gene therapy are categorized into non-viral (6) and viral. Non-viral methods of gene delivery include cationic and cholesterol-containing liposomes, peptide-lipid vectors, activated dendrimers (branched DNA-binding carbohydrates), bacteria, artificial chromosomes and artificial viruses (liposomes with viral components).
Virus-mediated gene therapy systems
The viral vectors used for gene transfer include retroviruses, adenoviruses, adeno-associated viruses, herpes simplex virus and flaviviruses (7, 8) . Each specific vector group has its own limitations for use in human gene therapy. For instance, the use of adenoviruses is hampered by their induction of immune responses in the host and their inability to stably integrate into the host genome. The lack of the stable integration has also been a problem when using herpes simplex virus-based vectors. Adenoassociated viruses, although capable of integration into genomic DNA, depend on "helper" viruses (e.g. adenovirus or herpes simplex virus) for productive infection (9) . The major drawback of the commonly used onco-retroviral vectors is their inability to transduce non-dividing cells (10) .
The family Retroviridae comprises a large group of viruses that infect primarily vertebrates, although infection of other animals has been described in a few instances (11) . A unifying feature of these viruses is that replication involves the process of conversion of the viral RNA genome into double-stranded DNA, hence the designation "retro" (backward) viruses. Members of the Retroviridae family are classified into three subfamilies: (i) oncovirinae, which includes the oncogenic retroviruses and closely related non-oncogenic viruses; (ii) spumavirinae, whose members establish persistent infections in the absence of clinical disease and cause a characteristic cytopathic effect in vitro; and (iii) lentivirinae, a group of viruses that are associated with slow, progressive diseases affecting the immune system (7).
Lentiviruses include a variety of primate (e.g. human immunodeficiency viruses [HIV-1 and 2], and simian immunodeficiency viruses [SIV] ) and non-primate viruses (e.g. maedi-visna virus [MVV] , feline immunodeficiency virus [FIV] , equine infectious anemia virus [EIAV] , caprine arthrithis encephalitis virus [CAEV] and bovine immunodeficiency virus [BIV] ) viruses. The ability to integrate into the genome of non-dividing cells (12) (13) (14) makes lentiviruses particularly attractive in human gene therapy. Examples of non-dividing cells which are potential targets for gene therapy include hepatocytes, neurons, hematopoietic stem cells, myocytes and macrophages. Lentiviruses which are currently used as the basis for development of gene therapy vectors are HIV-1 and 2, SIV, FIV, EIAV and CAEV.
THE BIOLOGY OF LENTIVIRAL INFECTION
The biology of lentiviral infection has been reviewed extensively elsewhere (7, 15, 16) . This part of the chapter will provide an overview of the organization of lentiviral genome and life cycle with the emphasis on those aspects which are relevant to gene therapy.
Molecular aspects of lentiviral infection
Lentiviruses possess a complex genome. In addition to the structural genes (gag, pol and env) common to all retroviruses, lentiviruses also contain a plethora of regulatory and accessory genes involved in modulation of viral gene expression, assembly of viral particles and structural and functional alterations in the infected cell (table 1) .
Replication, integration and packaging of lentiviruses are mediated, in part, by cis-acting RNA or DNA sequences, which do not encode proteins (table 1) . Most of such cis-acting elements are essential in the design of lentiviral vectors and are usually included in the transfer element (i.e., the part of a retroviral or lentiviral vector which will integrate in the host cell genome and which encodes the gene of interest).
The trans-acting viral elements encode three groups of proteins: structural, regulatory and accessory. Lentiviral vectors are defective for replication and are, therefore, unable to produce progeny. Thus, only the early steps (attachment, entry, reverse transcription and integration) of the lentivirus life cycle must be maintained in a lentiviral vector. Since early steps do not depend on viral protein synthesis, most trans-acting genes can be excluded so that the transfer vector only encodes the gene(s) of interest. Elimination of the trans-acting genes renders the transfer vector replication-defective. Transacting genes are provided in trans by a packaging plasmid.
Structural genes of HIV-1
HIV-1 encodes three structural genes: gag, pol and env. The product of gag is translated from unspliced mRNA as a precursor protein and cleaved into the following protein subunits: matrix (MA), essential for virion assembly and infection of non-dividing cells (17, 18) ; capsid (CA), which forms the hydrophobic core of the virion and is essential for virion assembly and maturation; nucleocapsid (NC), which coats viral RNA stochiometrically and remains tightly associated with viral RNA in virions; and several additional polypeptides of small size and unknown function, such as p1, p2 and p6.
The pol gene encodes three enzymes required for viral replication: protease (PRO), reverse transcriptase (RT) and integrase (IN). PRO cleaves Gag and Gag-Pol polyproteins which are essential for the maturation of the virion. RT contains three enzymatic activities: RNAdependent DNA polymerase, RNAase H and DNAdependent DNA polymerase.
In addition, the pol genes of certain non-primate lentiviruses (FIV, CAEV, MVV, EIAV) encode deoxyuridine triphosphatase (dUTPase), an enzyme that decreases intracellular concentration of dUTP and thus limits misincorporation of deoxy-uracil into viral DNA (19) (20) (21) (22) . It is thought that the presence of dUTPase in these viruses decreases the mutation rate by reducing incorporation of deoxy-uracil into DNA during reverse transcription (19) .
The env gene is essential for viral binding and entry into the host cells. It encodes the precursor glycoprotein, gp160, which is cleaved into a surface moiety, gp120 (SU), and a transmembrane moiety, gp41 (TM). The surface glycoprotein is required for binding to cellular receptors, whereas the transmembrane glycoprotein is responsible for the fusion with the cellular membrane.
The regulatory genes tat and rev encode transactivator proteins essential for viral replication. The tat gene is present in all lentiviruses except FIV (23) . The rev gene is present in all lentiviruses. Doubly spliced viral mRNAs encoding Rev, Tat and Nef proteins are the first ones to be synthesized de novo following viral integration. Once synthesized, Rev and Tat augment production of viral mRNAs.
Rev contains a nuclear export signal (NES) and allows nuclear export of unspliced and singly spliced mRNAs that encode viral structural proteins. In the absence of Rev the only mRNAs detected in the infected cells are doubly-spliced ones. Two interactions of Rev are required for attaining this function. First Rev interacts with the Rev responsive Element (RRE) overlapping the env coding sequence. Second, Rev interacts with the nuclear pore proteins, nucleoporins. Rev, therefore, acts as a shuttle between the nucleus and cytoplasm, mediating the nuclear export of the RRE-containing viral mRNAs.
Since it is absolutely essential for viral replication, Rev has been a target for a number of anti-HIV gene therapeutic strategies (24, 25) including protein-and RNA-based strategies (26) (27) (28) ). In HIV-1-based vectors, the presence of rev is essential unless its function is accomplished by an alternative genetic element. Thus, Rev/RRE can be substituted with the cis-acting 219-nucleotide constitutive transport element (CTE) from the Mason-Pfizer monkey virus (MPMV) that has been shown to allow Rev-independent HIV-1 replication (29).
The viral protein Tat upregulates viral transcription at the level of elongation via interaction with the Tat activation region (TAR) located at the 5' end of all viral mRNAs. In addition to interacting with TAR, the binding to the cyclin-dependent kinase (Cdk)-Cyclin T complex has also been shown to be required for the activity of Tat (30). The potential gene therapy approaches involving Tat include trans-dominant negative mutants (31, 32) and TAR decoys (28).
In gene therapy vectors, requirement for Tat can be overcome by including a strong constitutive promoter, such as the human cytomegalovirus immediate early (HCMV-IE) enhancer/promoter into the transfer vector (33, 34).
The accessory genes include vif, vpr, vpu and nef. These genes were named accessory because they are nonessential for virus replication in cell culture (35).
Vif (virion infectivity factor) has been identified in all lentiviruses except EIAV. Vif is not essential for HIV-1 replication in permissive cells such as HeLa-CD4 or SupT1 (36, 37). However, it is necessary for production of infectious virions by cells that are natural targets for infection, including CD4-positive T-lymphocytes, macrophages, and H9 cells (38-40). A recent study has suggested that nonpermissive cells contain an endogenous inhibitor of HIV-1 production that is overcome by the virus-encoded Vif protein (41). The potential role of Vif in an HIV-1-derived vector was directly examined and it was concluded that vif is dispensable (42, 43). It may be inferred that the vector producing cells utilized in these studies (42, 43) may have a "permissive" phenotype with respect to Vif, and that the presence of Vif in a replicationdefective vector may not be necessary because target cells will not produce viral progeny.
Vpr (viral protein R) is a virion-associated protein present only in primate lentiviruses. The first function of Vpr to be documented was its ability to act as a weak transcriptional transactivator of the viral LTR (44, 45). In addition, HIV-1 Vpr participates in viral infection of non-dividing cells (12, (46) (47) (48) . Vpr has also been shown to cause cell cycle block in G2 and apoptosis (49-54). Despite its role in the transduction of non-dividing cells, vpr is not required in an HIV-1 packaging construct (42, 43), due largely to the existence of the other mechanisms (MA and IN) allowing the nuclear transport of preintegration complexes.
The vpu gene encodes a cytoplasmic viral protein and present exclusively in HIV-1. It promotes degradation of CD4 in the endoplasmic reticulum of target cells. Degradation of CD4 allows transport of Env to the cell surface and its incorporation into virions. Vpu is able to stimulate the release of viral particles from certain types of cells including T-lymphocytes, HeLa cells and colonic carcinoma SW480 cells (55, 56). Another function of Vpu is to downregulate the expression of MHC I molecules on the surface of infected cells (57) . This prevents recognition by cytotoxic T-cells. Despite its multiple functions, vpu is a dispensable gene in vector system as its exclusion from the vector does not not seem to influence the properties of the vpu-negative viral particles (42, 43).
The nef gene is only found in primate lentiviruses and it is essential for viral infectivity in vivo, but not in vitro (58) . Nef reduces interactions between Env and intracellular CD4 by inducing internalization and degradation of CD4 (59) . Nef also downregulates cell surface expression of MHC I molecules and protects infected cells from killing by cytotoxic T-lymphocytes (60) . Nef was also shown to enhance the infectivity of viral particles, independently of the effects on CD4 (61, 62) . This Nef-dependent enhancement of infectivity occurs at the level of proviral DNA synthesis, early in the infectious cycle (63) (64) (65) . However, later studies demonstrated that the dependence on Nef for achieving optimal infectivity in a vector system could be overcome by the use of VSV-G glycoprotein-pseudotypes (66) . This observation suggested that Nef only increases infectivity of virions entering the cells by direct fusion with the cellular membrane, but not via receptor-mediated endocytosis. These results are in agreement with other studies which directly demonstrate that Nef is entirely dispensable in lentiviral vectors when pseudotyed with VSV-G (42, 43).
Other open reading frames found in the genomes of lentiviruses include orf2 of FIV, S2 orf of EIAV and tmx, vpy and vpw of BIV. Orf2 is a transcriptional transactivator needed for replication of FIV in primary Tlymphocytes (23) . The function of S2 orf is still unknown. S2 orf is not an essential gene, however, since it is not required for viral infectivity or replication (67) . The functions of the tmx, vpy and vpw genes of BIV are not known. It has been speculated that they encode proteins which are analogues of Nef, Vpr/Vpx and Vpu of other lentiviruses (68).
Molecular determinants of infection of nondividing cells
Among retroviruses, the ability to integrate into the genome of non-dividing cells is unique to lentiviruses. Three molecular elements appear to determine infection of non-dividing cells by HIV-1: MA, IN and Vpr (12, 17, 18, 69) . These proteins utilize the cellular nuclear import machinery to target the pre-integration complex (PIC) to the nucleus.
The cellular nuclear import system consists of the cytosolic proteins, importin-α and importin-β and nuclear pore proteins called nucleoporins. Importin-α carries a binding site for nuclear localization sequences (NLS). When NLS-containing proteins bind to importin-α, conformational changes occur that permit interaction with importin-β. Importin-β then targets the complex to the nuclear pore by binding it to the nucleoporins (70).
Both MA and IN carry conventional NLSs and utilize the importin-α/β pathway for nuclear transport. Vpr, on the other hand, does not contain a canonical NLS and acts as an importin-β analogue (71) interacting with both importin-α and nucleoporins.
Moreover, the interaction of Vpr with importin-α increases the affinity of importin-α for the NLSs of MA and IN and, therefore, enhances nuclear import of the PIC into non-dividing cells.
In addition to participating in the nuclear transport of PICs, Vpr was shown to induce cell cycle arrest in G2 and apoptosis (See section 3.1.1 above). From a theoretical standpoint, an ideal lentiviral vector would encode the former function of Vpr (nuclear transport of PICs) but not the latter (cell cycle arrest and apoptosis). This was possible in a SIV-based packaging system due to the segregation of the function into vpx (nuclear transport) and vpr (cell cycle arrest). As predicted, a vector expressing SIVmac vpx, but not SIV mac vpr was fully infectious in non-dividing cells (72) . In practice, however, it has been proposed that Vpr may be entirely dispensable in a lentiviral vector without dramatically affecting infectivity of non-dividing cells (42, 43).
Life cycle of lentiviruses
The lentiviral life cycle is similar for all the members of the Retroviridae family and consists of the steps outlined below (sections 3.2.1. to 3.2.5.).
Attachment and entry
The interaction of the lentivirus with the target cell occurs via binding of the viral envelope glycoprotein to a specific receptor on the cell membrane, which defines the cellular target for the virus (viral tropism). Primate lentiviruses infect helper Tlymphocytes, macrophages, microglial, dendritic and Langerhans cells by interacting with CD4 and one of several chemokine receptors, most frequently CCR5 or CXCR4 (73) (74) (75) (76) . FIV utilizes feline homologues of the CD9 receptor (77) and CXCR4 (78) to infect CD4+ and CD8+ T lymphocytes, B-lymphocytes, platelets, monocytes, astrocytes, microglia and dendritic cells.
Once bound to the cellular receptor, the viral membrane undergoes fusion with the cellular membrane (79) . After virion-bound matrix and capsid proteins disassemble and the nucleoprotein complex is delivered into the cells, reverse transcription begins in the cytoplasm.
One property common to most retroviruses is the ability to form pseudotypes, retroviral particles which incorporate a heterologous envelope glycoprotein (80) (81) (82) . The envelope glycoprotein G of the vesicular stomatitis virus (VSV-G) is commonly used to create such pseudotypes as it provides two major advantages to the development of the gene delivery vectors. First, entry of VSV into the target cell occurs via binding to ubiquitous phospholipid components of the cell membrane: phosphatidylinositol, phosphatidylserine and GM3 ganglioside (83) . This mode of entry provides VSV (and most retroviruses pseudotyped with VSV-G) a broad host range which includes non-mammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera (84).
Second, the VSV envelope forms highly stable viral particles. Owing to this increased stability, efficient concentration of the vectors can be achieved by ultracentrifugation (84, 85) . Concentration of vectors leads to increased titers by about 2 orders in magnitude.
The first VSV-G pseudotyped retroviral vector in which the only element from VSV was the glycoprotein G was described by Burns and coworkers (84) . This vector contained the core and genome of the murine leukemia virus. When concentrated by ultracentrifugation, this vector exhibited titers of approximately 10 9 infectious units per milliliter (IU/ml), a titer which would be difficult to achieve when using the homologous (MuLV) envelope glycoprotein.
Reverse transcription
Reverse transcription leads to the synthesis of double-stranded, linear DNA from a single-stranded RNA template using cellular tRNA as a primer. The enzyme that catalyzes this step in viral replication, reverse transcriptase, has low fidelity due to the lack of proofreading ability and therefore, is partly responsible for the high variability of the viral genome.
Integration
Once synthesis of the linear viral DNA is complete, viral integrase performs specific cleavages at the 5' and 3' termini and catalyzes integration into the host genome. Integration is essential for retroviral gene expression (86, 87) and allows the provirus to become a permanent genetic element in the host.
Accessibility of different regions of the genome for avian leukosis virus (ALV) was directly studied by Withers-Ward and collaborators (88) .
This study concluded that there were no preferences for integration within the genome of avian cells, although local differences in DNA structure (as evidenced by DNAse I susceptibility) may affect the integration specificity. Integration for other retroviruses is expected to follow a similar pattern, although this remains to be directly tested (88) . Attempts to manipulate the site of retroviral integration have achieved a certain degree of success (89) but this strategy will need further development.
Transcription and viral protein synthesis
Early transcription from the provirus results in the production of doubly spliced mRNAs encoding Rev, Tat and Nef. Translation of mRNA leads to accumulation of Tat and Rev. Together, Tat and Rev induce shift to "late" transcription mode whereby unspliced and singly spliced RNA species are primarily produced. Unspliced and singly spliced mRNAs encode structural genes necessary for the production of viral progeny.
Virion assembly and release
The viral RNA and structural proteins are packaged into viral particles and released by budding at the plasma membrane. After the polyproteins, Gag and Gag/Pol, are cleaved by the viral protease, mature particles become fully infectious. In currently available lentiviral vectors, gag/pol are not present in the transfer construct, but are provided in trans by a packaging construct (72, 90) . Thus, once the transfer vector is integrated in the host cell, its inability to direct production of gag/pol ensures that there will be no subsequent viral progeny.
LENTIVIRAL VECTORS

Applications of lentiviral vectors
The applicability of a safe lentiviral vector in human disease is broad because (1) the host range of lentiviruses can be virtually unlimited when using vesicular stomatitis G glycoprotein (VSV-G) to produce envelope pseudotypes; (2) many relevant targets for gene therapy are non-dividing cells (neurons, hepatic cells, hematopoietic stem cells, myocytes); and (3) the stability of the transgene is potentially indefinite due to chromosomal integration. Lentiviral vectors offer potential for treatment of a wide variety of syndromes (table 2) , including genetic/metabolic deficiencies, viral infection and cancer. Inherited genetic defects such as adenosine deaminase deficiency, familial hypercholesterolemia, cystic fibrosis, mucopolysaccharidosis type VII, types I and II diabetes, classical phenylketonuria and Gaucher disease may be overcome by lentiviral vector-mediated gene therapy because they constitute single-gene deficiencies for which the involved genes are known. In fact, an HIV-1-based vector was constructed that successfully delivered the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) into human undifferentiated bronchial epithelium cells derived from the CF patient implanted into nude mice. Expression of CFTR resulted in functional correction of the CF defect after maturation of the airway epithelium (91) .
Certain types of cancer may benefit from the use of lentiviral vectors. Hypoxia and lack of vascularization lead to the generation of tumor cells which exhibit limited or no proliferation. Partly because of the lack of growth, these cells are highly resistant to genotropic agents. A lentiviral vector may prove to be a useful vehicle for delivery of a "lethal" gene (such as herpes virus thymidine kinase) to quiescent tumor cells.
Viral diseases may also constitute appropriate targets for lentiviral gene delivery. In particular, a number of gene therapy approaches have been proposed for the treatment of HIV infection (see section 171.132.149.166.). For some of these strategies, phase I studies have recently begun in humans. Preliminary studies have dealt with defective murine oncoviruses for delivery of anti-sense RNAs, ribozymes and trans-dominant proteins against HIV replication.
Elements of a prototypical lentiviral vector
Contemporary lentiviral vectors are produced via a transient expression system that consists of three genetic elements: transfer construct, packaging construct and Env expression construct (72, 84, 90) . The transfer construct contains retroviral cis-acting elements and the gene(s) of interest. The packaging construct directs expression of viral structural proteins, except for the envelope. Proteins expressed by the packaging construct (Gag/Pol, predominantly) form the capsid and polymerase components, and recognize specific cis-acting sequences in the retroviral RNA genome and its reverse-transcribed DNA products (92) . This recognition leads to reverse transcription and integration. The envelope construct typically contains a heterologous envelope (e.g. vesicular stomatitis virus glycoprotein G [VSV-G]). The three expression constructs are maintained in the form of bacterial plasmids and can be transfected into mammalian cells to produce replication-defective virus stocks (72, 90 ).
An example of such a prototypical lentiviral vector (figure 1) was described by Naldini and collaborators (90) . In this vector system, the transfer construct contained a reporter gene (either β-galactosidase or luciferase) under control of an internal CMV promoter and the cis-acting sequences of HIV-1 including LTRs, splice signals, about 350 base pairs of the gag gene and the RRE. The packaging construct contained all the viral proteins (except Env and Vpu) whose expression was driven by the HCMV promoter. All the cis-acting sequences required for packaging (Ψ), reverse transcription (PBS, PPU) and integration (att) were deleted from the packaging construct. In addition, the 5'LTR was substituted by the HCMV-IE promoter, and the 3' LTR was substituted by a polyadenylation signal from the insulin gene. and host range, as they were able to infect a wide variety of cells including CD4+ and CD4-human cells and murine cells. The viral titers yielded by such systems were less than 2x10 5 IU/ml. Subsequent studies (95, 96) used similar two-plasmid systems to derive the HIV-1-based vectors which were used to study envelope dependence of the viral hostrange and biological consequences of HIV-1 integration.
These vectors were produced by the cotransfection of the env-defective HIV-1 bearing the firefly luciferase reporter gene in place of nef with a vector expressing the envelope glycoproteins of various HIV-1 strains or amphotropic MLV envelope. The infectivity of the resulting vectors was tested in a variety of cells of primate (e.g. human and simian) and non-primate origin (e.g. murine), and their tropism correlated with that of the virus that gave rise to the env gene.
Buchschacher and Panganiban (97) separated the viral genome into two constructs: a packaging construct and a transfer construct. The packaging construct expressed all the proteins, except Env. Env would be required in trans for production of infectious viral particles. The transfer construct contained a reporter gene and the cis-acting sequences required for packaging, reverse transcription, integration and expression of viral genes. Inclusion of about 700 bp located at the 5' end of gag into the transfer construct increased the transduction efficiency of the viral particles. These vectors had low titers of about 10 2 IU/ml.
The use of low-titer vectors for gene therapy can be problematic because of the low transduction efficiency in certain cell types such as hematopoietic cells (98, 99) . By using the vesicular stomatitis virus envelope glycoprotein G (VSV-G), vector stocks can be concentrated to titers of about 10 7 IU/ml (85, 100).
First generation of HIV-1-based vectors for gene therapy
Knowledge about the roles of cis and transacting elements of HIV-1 led to the first generation of HIV-1-based vectors intended as a gene delivery vehicles (101). Parolin and coworkers developed a packaging system which segregated viral structural genes into two different plasmids. One of the packaging plasmids expressed gag, pol, tat and vif under the control of the HCMV-IE promoter.
The second packaging construct expressed env and rev. In both plasmids, the packaging signal (region between the 5' splice donor site and the initiation codon of gag) was deleted and the 3' LTRs were replaced with the polyadenylation sequence from simian virus 40. The transfer constructs contained cis-acting sequences required for efficient transduction and encoded the neomycin phosphotransferase (neo R ) reporter gene under control of ecotropic MLV LTR. These vectors had titers of approximately 10 2.2 IU/ml. Despite such low titers, these studies provided the conceptual basis for the subsequent development of more efficient vector systems.
Naldini et al. (90) described a similar vector system that also utilized three constructs: transfer, packaging and envelope. These vectors yielded titers of about 10 5 IU/ml when pseudotyped with the MLV envelope and about 5X10 5 IU/ml when pseudotyped with VSV-G. VSV-G pseudotyped HIV-1 vectors successfully transduced growth-arrested HeLa cells and rat 208F fibroblasts and were able to mediate stable in vivo transfer into adult rat neurons. However, significantly lower transduction efficiencies were observed when same cells were transduced with MLV pseudotyped HIV-1 vectors. Kafri et al. (102) constructed a subsequent vector lacking Vpr and Vif proteins, which efficiently delivered the green fluorescent protein (GFP) gene in vivo into rat liver and muscle. Long-term expression of the transgene was observed in the transduced tissues (more than 5 months in the liver and more than 8 weeks in the muscle). No immune response was elicited by the transduced cells, thus making subsequent administration of vector possible.
Role of accessory genes in transduction by HIV-1-derived vectors.
Pathogenesis by lentiviruses is largely attributed to the action of the accessory genes. Thus several groups tested the possibility that accessory genes may be eliminated from lentiviral vectors (42, 103, 104). This was accomplished by creating various packaging constructs with deletions in all accessory genes. Infectious titers and the ability to transduce non-dividing cells were then measured.
High-titer (2x10 6 IU/ml) retroviral particles deleted in all accessory genes (vif, vpu, vpr, nef) were produced from a system developed by Zufferey et al. (42) . These vectors were able to mediate efficient in vitro transduction into dividing and growth-arrested 293T cells and in vivo delivery into adult rat neurons. Although the presence of Vpr was dispensable for transduction of neurons, about 50% reduction was observed when using Vpr(-) vector to transduce macrophages. No significant differences in vector properties were attributable to nef, vif or vpr.
Other groups (103, 104) used multiply attenuated vectors to transduce human hematopoietic stem cells, cardiac myocytes and post-mitotic rat cerebellar neurons. In agreement with the studies by Zufferey et al. (42) , these studies concluded that viral accessory genes (vpr, vif, vpu and nef) are dispensable for efficient gene delivery into non-dividing cells.
Elimination of Tat and Rev functions.
Using a conventional three-plasmid expression system Dull and co-workers (34) created a vector whose packaging construct was reduced to the gag, pol and rev genes. A defect in tat function was created by introducing two mutations into the tat gene: mutation of the translational initiation codon and insertion of a linker producing a translational stop at codon 46. The requirement for tat was overcome by replacing the U3 region of the 5' LTR with the HCMV enhancer/promoter. Surprisingly, the tat-deficient phenotype did not affect either the titer or transduction efficiency of the viral particles. These vectors had a titer of about 5x10 6 IU/ml and efficiently transduced rat neurons in vivo. Furthermore, vectors with improved biosafety features (34) were constructed by deleting the rev gene from the packaging construct and providing Rev in trans on a separate expression plasmid. Co-transfection of the tat-free transfer construct, VSV-G construct, a plasmid expressing gag-pol and a plasmid expressing rev, yielded high-titer viral particles, demonstrating the efficiency of such a splitgenome packaging system.
Gasmi and co-workers (105) chose a similar approach to create a safe gene therapy vector: they deleted rev from the packaging construct thus reducing it to gag, pol and tat. The requirement for Rev, an essential player in HIV-1 replication, was bypassed by replacing RRE in the packaging construct with the constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV). Although the CTE-containing vector allowed Rev/RRE-independent expression of gag, its titer on 293T cells was 10-fold lower than that of an RRE-containing vector. This finding indicated that CTE could only partially substitute for the function of Rev/RRE, and that vectors could be generated most efficiently if Rev and RRE were present in the packaging construct.
Conditionally replicating HIV-1-based vectors
The use of Tat and Rev may allow construction of conditionally replicating or regulated gene therapy vectors. A vector was constructed by Buchschacher and Panganiban (97) where the reporter gene was placed upstream of the major 5' splice donor and, therefore, expressed directly from the LTR in a Tat-dependent fashion. Such a vector was able to propagate in HeLa-T4 cells expressing Tat, but not in parental HeLa T4 cells. It was speculated that Tat-dependent vectors can be used in the future to carry anti-HIV genes and, therefore, to treat individuals previously infected with HIV.
Conditionally replicating vectors (crHIV) capable of inhibiting HIV-1 infection were constructed by Dropulic et al. (106)
. These vectors contained deletions in all of the viral genes and encoded a U5-specific hammerhead ribozyme that selectively cleaved HIV-1 RNA. The U5 region of crHIV RNA was rendered ribozyme-resistant by substituting critical oligonucleotides at the cleavage site. Such vectors were only able to replicate in the presence of live HIV-1 and, when co-transfected into Jurkat cells, inhibited viral replication.
HIV-1 packaging cell lines
The lentiviral vectors described above were produced by transient transfection of multiple plasmids. Although relatively high titers of RCR-free virus can be produced by this method, transient transfection systems have certain drawbacks. First, the infectious titer of the transiently produced vectors is subject to high batch-tobatch variability. Second, the volume of vector particles produced by this method is limited. Thus, stable packaging cell lines have been constructed for large-scale production of replication-defective vectors. These cell lines provide in trans all viral proteins required for assembly of viral particles.
In the HIV-1 packaging cell line designed by Corbeau et al. (107) , the major packaging site, including 37 nucleotides between the major splice donor site and the beginning of gag was deleted from HIV-1 genome. The resulting packaging plasmid (Ψ) was co-transfected with a plasmid containing the neo R gene into HeLa cells and, after selection with G418, a resistant clone named Ψ422 was isolated. Although, the Ψ422 cell line was able to synthesize HIV-1 proteins, virions produced by these cells were noninfectious due to the inability of the genomic RNA to be packaged. To assess gene transfer, Ψ422 cells were transfected with an HIV-1-based transfer vector (101) . This vector contained sequences responsible for packaging including the first 653 nucleotides of gag and the RRE. Viral particles produced in this manner were free of RCR and were capable of transducing CD4+ HeLa-T4 cells, but the titer was relatively low (approximately 5x10 4 IU/ml).
A potential problem with the type of packaging cell line described by Corbeau et al. (107) is that a single recombination event between the HIV-1 sequences in the packaging cells and the transfer vector could lead to the production of the RCR. To overcome this problem, a split genome approach was developed by Srinivasakumar et al. (108) . To create the packaging cell line, CMT3-COS were stably transduced with five separate plasmids. One of the plasmids expressed gag-pol and vif and the remaining plasmids expressed one of the other genes, including env, nef, tat or rev. The 5' sequences required for packaging were deleted from each plasmid. When transfected with a transfer vector, the packaging cells produced helper-free viral particles able to infect HeLa-CD4 cells with titers of up to 10 4 IU/ml.
In addition to providing desired safety features, split genome systems allowed an assessment of the effects of individual viral proteins on vector titer by introducing one gene at a time. Thus, in the vector system developed by Srinivasakumar and coworkers, the presence of either Tat or Nef in the packaging cells increased the viral titers about 5-to 10 fold, whereas the presence of Rev was absolutely essential for vector production (108) . To circumvent the requirement for Rev and RRE, the constitutive transport element from MPMV was introduced into packaging plasmids replacing RRE. In addition, rev was eliminated from the producer cells. Contrary to the results reported by Gasmi et al. (105) , the titers of the vectors produced by the above Rev-independent packaging cells were similar to those obtained using a Rev-dependent system.
The constitutive expression of HIV-1 proteins (e.g. Vpr and protease) is highly cytotoxic. To overcome this problem, inducible packaging cell lines were developed (109) (110) (111) . These packaging cell lines produce viral proteins only after the removal of tetracycline from the culture medium and yield low-titer (0.6-2 x10 3 IU/ml), RCR-free viral particles upon transfection with an HIV-1 transfer vector.
Packaging cell lines have one common drawback: they produce low vector titers when compared to transiently transfected producer cells. To address this problem, Kafri and co-workers (111) developed a tetracycline-inducible HIV-1 packaging cell line that produced VSV-G pseudotyped vectors. In this system, the VSV-G cDNA and an HIV-1 packaging cassette, which included all HIV-1 genes except env, were cloned into tetracycline-inducible vectors and stably transfected into the SODk0 cell line.
The resulting HIV-1 vectors displayed a titer of greater than 10 6 IU/ml and could be concentrated by ultracentrifugation to 10 9 IU/ml. Moreover, these vectors were capable of transducing growth-arrested HeLa cells, human embryonic fibroblasts, and adult rat neurons in vivo.
Safety issues regarding design of lentiviral vectors
The most urgent issue regarding the safety of lentiviral vectors is the potential for recombination leading to replication-competent retrovirus (RCR), also referred to as "helper". Generation of helper virus in preparations of replication-defective vectors has been documented in numerous instances involving oncoviruses (112) (113) (114) (115) (116) . In later generations of vectors in which viral protein-coding regions were split in the packaging cells, the frequency of recombination leading to helper was decreased, but not eliminated (117) . Helper virus has the potential for inducing pathogenesis as demonstrated by studies in which monkeys were infused with transduced bone marrow cells after ablation of endogenous marrow with gamma irradiation (112) (113) (114) . In these studies, helper virus gave rise to lymphoma in monkeys.
Contemporary lentiviral vectors were made virtually helper-free by segregating vector components into three plasmids (see section 4.2.).
Another important safety issue involves the activation of the cellular proto-oncogenes by the inserted provirus (7) . The genetic elements that can cause such insertional activation include: promoter, enhancer and the poly(A) site of the virus. For example, leukemia viruses (e.g. ALV) have been shown to induce lymphomas via the activation of the c-myc expression by the promoter of the provirus (118, 119) . Theoretically, insertion of HIV-1 LTR's might lead to transcriptional activation of heterologous genes. However, this has not been yet demonstrated. To address the issue of insertional activation, self-inactivating (SIN) vectors were developed (120) (121) (122) . The same principles were applied to recent lentiviral vectors (33, 123) . For example, self-inactivating vector described by Miyoshi et al. (33) was constructed by deleting 133 bp in the U3 region of the 3' LTR including the TATA box and the binding sites for Sp1 and NF-kB. This deletion not only minimizes expression of vector RNA, but it may also be useful in preventing the insertional activation of cellular protooncogenes by the integrated provirus.
Vectors based on lentiviruses other than HIV-1
The use of HIV-1-based vectors in human gene therapy faces a number of obstacles due mainly to the fact that HIV-1 is a deadly pathogen. Thus, the possibility of the appearance of RCR in preparations of HIV-1-derived vectors, though remote, has spurred efforts to develop vectors based on other lentiviruses.
HIV-2-based vectors
HIV-2-derived vectors have potential advantages over HIV-1-derived ones such as decreased pathogenicity and segregation of the genes responsible for infection of non-dividing cells (vpx) and growth arrest (vpr). Cis-acting regions of HIV-2 responsible for genomic encapsidation were defined by Poeschla et al. (124) who found that deletion of 61 nucleotides from the region between the major 5` splice donor and the gag initiation codon prevented HIV-2 packaging and replication, but not expression of viral genes. The packaging plasmid was derived from the apathogenic, HIV-2 kr , virus by deleting the 61-nucleotide fragment and replacing 3` LTR with the bovine growth hormone polyadenylation signal. Transient co-expression of the packaging construct with VSV-G and a transfer vector bearing lacZ yielded titers of 10 6 IU/ml before concentration and 10 8 IU/ml after concentration. These viral particles were able to efficiently transduce human T and monocytoid cell lines, growth-arrested HeLa cells, human macrophages, NTN2 neurons and human hematopoietic progenitor cells.
Moreover, Poeschla et al. used their packaging plasmid to develop an HIV-2 packaging cell line, which was capable of stable expression of HIV-2 structural proteins, but did not produce replication-competent virus (125).
RNA pseudotypes
Retroviral particles which package a heterologous genomic RNA are referred to as RNA pseudotypes (126, 127) . RNA pseudotype vectors have the potential advantage or decreased risk for homologous recombination between the packaging and the transfer vectors.
Corbeau and coworkers (128) used the HIV-1 packaging cell line Ψ422 (see section 4.3.6.) and HIV-2 transfer vector. This HIV-1/HIV-2-based vector exhibited titers of 10 4 IU/ml and was capable of transducing nondividing cells such as primary human macrophages.
An HIV-1/SIV-derived vector was recently described by our laboratory (72) . It employs elements from HIV-1 and SIVmac1A11. SIVmacA11 is an infectious molecular clone which is apathogenic in rhesus macaques (129, 130) . In vitro, SIVmac1A11 infects human and rhesus peripheral blood lymphocytes and monocyte-derived macrophages. A vector in which the packaging construct is based on SIVmac1A11 retains the features of other lentiviral systems (i.e. integration and infection of nondividing cells) yet it may constitute a safe alternative to HIV-1-derived systems (72) .
The RNA-pseudotype between HIV-1 and SIVmac1A11 was produced by transient co-transfection of three vectors. The packaging construct was derived from SIVmac1A11 lacking packaging sequences and expressing all SIV genes, except env and vpr. The transfer construct was derived from HIV-1, including cis-acting sequences required for packaging, reverse transcription and integration. The envelope plasmid encoded the VSV-G glycoprotein. These vectors were free of RCR, displayed titers of 2.3x10 6 IU/ml and were able to transduce growth arrested HeLa cells, human T-lymphocytes, fetal mouse neurons and human peripheral blood macrophages. In contrast, these non-dividing cells were resistant to infection by the murine leukemia-based vector, LNCX-GFP.
FIV-based vectors
Because FIV is a non-primate lentivirus, it is thought that its pathogenic potential in humans is low. Furthermore, FIV does not productively infect human or other non-feline cells. To explore the possibility of creating an FIV-based vector with extended tropism which could be used for human gene therapy, Poeschla and coworkers (131) used a three-plasmid expression system. The packaging plasmid was constructed by replacing the 5L TR with the HCMV promoter and the 3` LTR by the bovine growth hormone polyadenylation signal. In addition, 875 nucleotides from the env gene were deleted. In the transfer construct, the U3 element within 5` LTR was replaced by fusing the HCMV promoter to the R repeat. A frameshift mutation was introduced into the gag gene and either lacZ or GFP reporter genes driven by the HCMV promoter were inserted. When co-transfected with VSV-G expression plasmid, viral particles were produced that exhibited titers of 10 5 IU/ml and were capable of transducing primary human macrophages and postmitotic NTN2 neurons. Most importantly, this work revealed that the 5` U3 region of FIV limits the productive phase of FIV replication in human cells and that the substitution of this region with the heterologous promoter (HCMV) allows the expression of FIV proteins in human cells.
Other potential lentiviral vectors
Other non-primate lentiviruses are under investigation for potential use as gene delivery vehicles. For example, replication-defective vectors based on Caprine Arthritis Encephalitis Virus (CAEV) were constructed by Mselli-Lakhal et al. (132) . The CAEVderived vectors were only tested on goat cells lines and had a low transduction efficiency (3-4 logs lower than that of the parental CAEV). Other potential candidates for use in human gene therapy include equine infectious anemia virus (6) and bovine immunodeficiency virus.
PERSPECTIVE
Since the inception of the Human Genome Project in 1990, considerable progress has been made in our understanding of human genetics (133) . More diseaserelated genes are being constantly identified and characterized widening the range of applications for human gene therapy.
This growing body of knowledge requires development of new and improved gene delivery vectors. Development of "helper" detection assays for lentiviral vectors will be necessary prior to testing in humans. A standard assay for detection of potential replicationcompetent virus generated in defective lentiviral systems does not exist at the moment. It will be crucial to the safety of novel gene therapy vectors to develop rapid and sensitive assays which will detect such recombinants in vector preparations. We envision that this may be accomplished by the use of indicator cells which show apparent cytopathic effects (syncytium formation) and/or express inducible reporter genes under the control of a lentivirus promoter. Development of a helper assay should be a concerted effort among many groups who specialize in the production and use of lentiviral vectors, so that detection conditions can be standardized.
The use of lentiviral vectors in humans will also require investigators to develop a large-scale production methods which are currently unavailable. Once these obstacles are overcome, the potential for modifications of the human genome using lentiviral vectors is virtually unlimited. 

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