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`Copyright c(cid:176) 2001 by Annual Reviews. All rights reserved
`
`GENE THERAPY: Promises and Problems
`
`Alexander Pfeifer and Inder M. Verma
`The Salk Institute, La Jolla, California 92037; e-mail: verma@salk.edu,
`apfeifer@ems.salk.edu
`
`Key Words gene transfer, viral vectors, gene therapy trials
`n Abstract Gene therapy can be broadly defined as the transfer of genetic material
`to cure a disease or at least to improve the clinical status of a patient. One of the
`basic concepts of gene therapy is to transform viruses into genetic shuttles, which
`will deliver the gene of interest into the target cells. Based on the nature of the viral
`genome, these gene therapy vectors can be divided into RNA and DNA viral vectors.
`The majority of RNA virus-based vectors have been derived from simple retroviruses
`like murine leukemia virus. A major shortcoming of these vectors is that they are not
`able to transduce nondividing cells. This problem may be overcome by the use of novel
`retroviral vectors derived from lentiviruses, such as human immunodeficiency virus
`(HIV). The most commonly used DNA virus vectors are based on adenoviruses and
`adeno-associated viruses. Although the available vector systems are able to deliver
`genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present
`viral vectors should be used only with great caution in human beings and further
`progress in vector development is necessary.
`
`INTRODUCTION
`
`Biologists will remember Monday, June 19, 2000, as an historic day. Flanking
`Bill Clinton, the 42nd President of the United States of America, were Francis
`Collins of the National Institutes of Health (NIH), leader of the publicly funded
`Human Genome project, and Craig Venter, CEO of Celera Genomics of Rockville,
`Maryland, to announce the near-completion of the sequencing of the human
`genome. Imagine: The entire 3 billion nucleotides of our genome are decoded—
`an impossible task just a few years ago. The estimate of the number of genes ranges
`from a low of 35,000 to a high of more than 100,000.
`What a bonanza for gene therapy. The science of gene therapy relies on the
`introduction of genes to cure a defect or slow the progression of the disease and
`thereby improve the quality of life. Therefore, we need genes. Suddenly, we have
`tens of thousands of them at hand. Though gene therapy holds great promise for
`the achievement of this task, the transfer of genetic material into higher organisms
`still remains an enormous technical challenge. Presently available gene delivery
`vehicles for somatic gene transfer can be divided into two categories: viral and
`
`1527-8204/01/0728-0177$14.00
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`nonviral vectors. Viruses evolved to depend on their host cell to carry their genome.
`They are intracellular parasites that have developed efficient strategies to invade
`host cells and, in some cases, transport their genetic information into the nucleus
`either to become part of the host’s genome or to constitute an autonomous genetic
`unit. The nonviral vectors, also known as synthetic gene delivery systems (45),
`represent the second category of delivery vehicles and rely on direct delivery of
`either naked DNA or a mixture of genes with cationic lipids (liposomes). In this
`review, we focus on viral vectors and highlight some examples of their use in
`clinical trials. A complete, constantly updated list of human gene therapy trials
`in the United States is available at the Office of Biotechnology Activities, NIH
`(http://www4.od.nih.gov/oba/rdna.htm).
`
`General Concept of Viral Vectors
`The first step in viral vector design is to identify the viral sequences that are
`required for the assembly of viral particles, the packaging of the viral genome into
`the particles, and the delivery of the transgene to the target cells. Next, dispensable
`genes are deleted from the viral genome to reduce patho- and immunogenicity.
`The residual viral genome and the gene of interest (also termed transgene) are
`integrated into the vector construct (Figure 1).
`Viral vectors can be divided into two general categories: (a) integrating vectors,
`capable of providing life-long expression of the transgene, and (b) nonintegrating
`vectors. Examples for integrating vectors are retroviral and adeno-associated virus
`(AAV)–derived vectors. The major nonintegrating vector currently employed is
`based on adenoviruses, and the viral DNA is maintained as an episome in the
`infected cell. Each of these vectors has specific advantages and major limitations.
`What, then, would be an ideal vector? We believe that it should fulfill the following
`requirements (147):
`
`1. Efficient and easy production: High-titer preparations of vector particles
`should be reproducibly available. The efficient transduction of cells within
`tissues is only possible if a sufficient number of infectious particles reaches
`the target cells. For the widespread use of viral vectors, facile production
`procedures have to be developed.
`
`¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡!
`Figure 1 Basic principal of viral vector design. (A) Structure of a generic viral
`genome. (B) Strategy of gene therapy vectors. The viral genome is separated into the
`packaging construct, which contains the viral sequences encoding proteins required for
`packaging of the vector genome and its replication. The vector construct contains the
`transgene and cis-acting sequences (hatched boxes) that are essential for encapsidation
`of the vector genome and for viral transduction of the target cell. (C) The vector
`and packaging constructs are expressed in the packaging cells, which produce the
`recombinant viral particles.
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`2. Safety aspects: The vector should neither be toxic to the target cells nor induce
`unwanted effects, including immunological reactions against the viral vector
`or its cargo. The latter carries not only the threat of eliminating the vector
`and/or the infected cells but also may lead to life-threatening complications,
`such as septic shock.
`3. Sustained and regulated transgene expression: The gene delivered by the
`viral vector has to be expressed in a proper way. Permanent or even life-long
`expression of the therapeutic gene is desired only in a minority of diseases
`(e.g., treatment of hemophilia). Controlled expression of the transgene in
`a reversible manner would be highly desirable in many cases (e.g., gene
`therapy for insulin-dependent diabetes mellitus).
`4. Targeting of the viral vectors: Preferential or exclusive transduction of spe-
`cific cell types is very desirable.
`5. Infection of dividing and nondividing cells: Because the majority of the cells
`in an adult human being are in a postmitotic, nondividing state, viral vectors
`should be able to efficiently transduce these cells.
`6. Site-specific integration: Integration into the host genome at specific site(s)
`could enable us to repair genetic defects, such as mutations and deletions, by
`insertion of the correct sequences. Thus, replacing defective gene expression
`by introducing foreign genes and cDNAs would be unnecessary.
`
`RNA VIRUS VECTORS
`
`RNA viruses are a large and diverse group of viruses (150) that have either a
`single-stranded or a double-stranded RNA genome. They can infect a broad spec-
`trum of cells, ranging from prokaryotes to many eukaryotic cells. Among the
`RNA-containing viruses, one group has attracted much attention as a gene de-
`livery vehicle: the Retroviridae (30). Retroviruses comprise a diverse family of
`enveloped RNA viruses and can be divided into two categories according to the
`organization of their genome: simple and complex retroviruses (29). All retro-
`viruses contain three major viral proteins: gag, pol, env [Figure 2, right; for review
`see (30, 160)]. Gag encodes the structural virion proteins that form the matrix,
`capsid, and the nucleoprotein complex. Pol codes for the essential viral enzymes
`reverse transcriptase and integrase. Env encodes the viral glycoproteins that are
`displayed on the surface of the virus. Moloney murine leukemia virus (MLV), a pro-
`totypic simple retrovirus, carries only a small set of genetic information, whereas
`the complex retroviruses like lentiviruses [e.g., human immunodeficiency virus
`(HIV)] contain additional regulatory and accessory genes. Initially, gene therapy
`vectors were developed from simple retroviruses. The lessons learned from simple
`retroviral vectors provided an invaluable basis for the development of vectors de-
`rived from complex retroviruses. Emerging vectors based on other RNA viruses,
`such as alphaviruses, are reviewed elsewhere (71).
`
`Annu. Rev. Genom. Hum. Genet. 2001.2:177-211. Downloaded from www.annualreviews.org
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`
`Figure 2 Retroviral lifecycle. (Left) Overview of the replication cycle of a prototypic retro-
`virus, MLV. (Right) Organization of the retroviral genome and its transition from RNA !
`DNA ! RNA during viral replication. The prototypic retroviral genome contains the gag,
`0
`pol, and env genes flanked by the R/U5 region at the 5
`end and the U3/R region at the 3
`end.
`Reverse transcription results in the proviral DNA that contains long terminal repeats (LTRs)
`at each end. The LTRs comprise U3, R, and U5 elements in the provirus. Transcription be-
`0
`0
`tween (not including) the 5
`U3 and the 3
`U5 regions generates the identical organization of
`the terminal domains as in the parental virus (top). A(n), polyA tail.
`
`0
`
`Retroviral Life Cycle
`Knowledge of the viral life cycle was crucial for the development of retroviral
`vectors. Following infection of the cell, the genomic RNA is reverse transcribed
`into linear double-stranded DNA by the virion reverse transcriptase (156). Reverse
`0
`end to
`transcription involves two jumps of the transcriptase enzyme from the 5
`0
`end of the viral template, causing a duplication of the sequences located at
`the 3
`the ends of the viral RNA. Thus, the viral DNA is significantly longer than the
`0
`0
`and 3
`ends. The resulting tandem repeats in the viral
`viral genome at both the 5
`DNA are termed long terminal repeats (LTRs) (Figure 2). Reverse transcription
`takes place in the cytoplasm and the viral DNA is translocated into the nucleus.
`Simple and complex retroviruses enter the nucleus of the host cell by two dif-
`ferent mechanisms: Nuclear entry of simple retroviruses can only occur when
`the nuclear membrane is disassembled and is, therefore, mitosis dependent. In
`
`Annu. Rev. Genom. Hum. Genet. 2001.2:177-211. Downloaded from www.annualreviews.org
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`contrast, lentiviruses can access the nucleus of nondividing cells by import through
`the nuclear pore [for review see (19)]. This has practical implications for the spec-
`trum of target cells that can be transduced by viral vectors derived from simple
`or complex retroviruses (see below). After entry into the nucleus, the viral DNA
`integrates into the host genome to form a provirus. The formation of a provirus is
`a unique genetic strategy: The DNA intermediate stage mimics a cellular gene and
`uses the host-cell machinery for gene expression. Therefore, the provirus requires
`cis-acting elements that control the host transcriptional machinery. Most of these
`elements are situated within the proviral LTRs. The complex retroviruses addi-
`tionally contain trans-acting factors that serve as activators of RNA transcription
`(e.g., HIV-1 Tat).
`The LTRs are divided into the U3, R, and U5 regions (Figure 2, right). The R
`0
`0
`LTR. In the 3
`LTR, the R
`region is defined as the transcription start site in the 5
`0
`-end processing (Figure 2, right panel). The U3 region that
`region is the target of 3
`0
`LTR contains the majority
`is found upstream of the transcription start site in the 5
`of cis-acting control elements. These elements regulate transcriptional initiation by
`0
`end
`the cellular RNA polymerase II. In addition, the sequence at the immediate 5
`of the U3 region contains the so-called att site that is necessary for integration. This
`0
`0
`LTR: The 3
`end of the U5 region contains
`sequence motif is also found in the 3
`0
`LTR contains the cis-acting
`an inverted copy of the att site. In addition, the 3
`0
`end of the
`control elements involved in posttranscriptional processing of the 3
`viral RNA (e.g., polyadenylation). Therefore, the transgene present in retroviral
`vectors should not contain a polyA signal sequence because this would lead to the
`0
`U3/R region with a polyA tail during transcription of the
`replacement of the 3
`vector RNA.
`The regulation of RNA processing differs between simple and complex retro-
`viruses and is an important aspect of gene therapy approaches that require unspliced
`transcripts for the expression of therapeutic genes. Retroviral RNAs are subject to
`0
`end, cleavage
`the same processing events as cellular RNAs: cap addition at the 5
`0
`end, and splicing [for review see (132)]. Simple
`and polyadenylation of the 3
`retroviruses regulate the cytoplasmic ratio of full-length versus spliced RNAs
`through cis-acting elements within the RNAs, whereas complex viruses encode
`proteins that regulate the transport and, presumably, the splicing of the viral RNA.
`For example, the Rev protein of HIV promotes the efficient transport of unspliced
`RNAs that contain Rev response elements from the nucleus to the cytoplasm (also
`see section on Lentiviral Vectors).
`After translation of the viral messages, the resulting protein products and the
`progeny RNA are assembled into viral particles that are released from the cell by
`budding of the plasma membrane (Figure 2, left).
`
`Retrovirus Vectors
`The majority of the retroviral vectors [see also (33, 100, 175)] presently used in
`gene therapy models are derived from MLV, and they were among the first viral
`vectors to be used in human gene therapy trials (18).
`
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`
`To generate retroviral vectors, all of the protein-encoding sequences were re-
`moved from the virus and replaced by the transgene of interest (Figure 3). The
`essential cis-acting sequences, such as the packaging signal sequences (9), which
`are required for encapsidation of the vector RNA, have to be included in the vec-
`tor construct. The viral sequences necessary for reverse transcription of the vector
`RNA and integration of the proviral DNA, the LTRs, the transfer RNA-primer bind-
`ing site, and the polypurine tract (PPT) [for a detailed description see (30, 100)]
`have to be present in the vector construct. Several modifications have been intro-
`0
`end
`duced into this basic retroviral vector system. For instance, inclusion of the 5
`of the gag domain leads to a 50–200-fold increase in vector titers, because of an
`increased efficacy of vector RNA encapsidation. MLV-based vectors carrying this
`extended packaging signal [nucleotides 215–1039 of gag (9)] are called 9C
`or
`C
`vectors (7, 9)
`gag
`0
`0
`LTR regions of the vector constructs have been subject to
`as well as 3
`The 5
`0
`LTR with the
`a number of modifications. Replacement of the U3 region in the 5
`immediate early region of the human cytomegalovirus (CMV) enhancer-promoter
`(Figure 3) resulted in an almost 100-fold increase (42, 116) in viral titers. The
`CMV/LTR hybrid has a high transcriptional activity, especially when introduced
`in the appropriate cell lines (42), e.g., human embryonic kidney (HEK), 293 cells.
`This cell line expresses the adenoviral E1 gene products (51) that superactivate
`the CMV promoter (48). The effects of this U3 modification are restricted to the
`0
`LTR of the provirus is derived
`packaging cells because the U3 region of the 5
`0
`end of the vector RNA (Figure 2). This hallmark
`from the U3 region of the 3
`of the retroviral life cycle is the basis for the development of transcriptionally
`silenced vectors, so-called self-inactivating (SIN) vectors (176, 181) (Figure 3).
`The SIN vectors were developed to cope with the problem of insertional activation
`of cellular oncogenes through the promoter and enhancer elements of the proviral
`LTR. The strategy of transcriptional inactivation of the provirus is based on the
`0
`fact that deletions of the promoter/enhancer sequences of the U3 region of the 3
`0
`LTR of the viral vector are carried over to the 5
`LTR during reverse transcription;
`in other words, the vector inactivates itself. The major drawback of the loss of
`transcriptional regulatory elements is a substantial reduction (10–100-fold lower)
`of viral titers. On the other hand, a partial deletion of the viral transcription control
`regions, with retention of the TATA box, results in only a partial abolotion of LTR-
`driven transcription (176). However, the use of SIN vectors may be necessary
`to avoid interference of the viral promoter and enhancer regions with internal
`promoters (see below) within the vector in the target cells (40). Taken together,
`the modifications of the vector LTRs significantly increased vector yields and
`improved biosafety of the retroviral vectors.
`
`REGULATION OF TRANSGENE EXPRESSION In the most simple vector design, the 5
`LTR of the integrated provirus drives the expression of the transgene (Figure 3).
`However, inclusion of an internal, heterologous promoter that drives transcription
`of the transgene in the target cells (Figure 3D) can either achieve an increase in
`
`0
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`Figure 3 Retroviral vector and packaging systems. Schematic representation of the MLV
`provirus. (B–E) The different retroviral vectors. (B) In the first generation of retroviral vectors
`based on MLV, the transgene replaces most of the viral sequences. In addition, it contains
`the necessary cis-acting factors, such as the LTRs and the packaging signal (9). (C) Modi-
`0
`fications of the 5
`LTRs result in an increase in vector yield (e.g., inclusion of a CMV/LTR
`0
`hybrid). SIN-mutations in the U3 region of the 3
`LTR (black triangle) improve the biosafety
`of the recombinant virus. (D) The latest generation of retroviral vectors incorporates inter-
`nal promoters (e.g., CMV) that drive transgene expression in the target cells. In addition, a
`0
`posttranscriptional element (WPRE) can be included 3
`of the transgene, which enhances ex-
`pression three- to fivefold. (E) Tetracycline(tet)-regulated expression system. In the absence
`of tet, transgene expression is activated by the tet repressor (tetR)-VP16 fusion protein, which
`binds to the tet operator (tetO) fused to a minimal human CMV promoter (mp). Both the trans-
`gene as well as the tetR-VP16 coding regions are included in one vector construct. Bicistronic
`expression is achieved by incorporation of an internal ribosomal entry site (IRES). (F, G)
`Retroviral packaging constructs. (F) First-generation packaging constructs contain deletion
`mutations of the packaging signal (9). Wild-type env (eco) can be replaced with amphotropic
`env (amph), resulting in a broadened host range. (G) Latest generation of split-genome pack-
`0
`aging system: gag/pol and env are encoded on separate plasmids. The 5
`LTR is replaced
`0
`with a strong promoter (e.g., CMV), and the 3
`LTR is replaced by a polyadenylation signal
`(polyA).
`
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`transgene expression and/or restriction of expression to a specifc cell type or tissue.
`In addition, this approach allows the incorporation of regulatable transcriptional
`elements that may be switched on and off via exogenous stimuli (Figure 3E).
`The regulatable systems used in viral vectors either are based on naturally
`occuring inducible promoters that exhibit tissue specificity or consist of chimeric
`systems, which contain pro- and eukaryotic elements from different organisms
`[for review see (2)]. Among the chimeric regulatable systems, the tetracycline-
`(tet)-regulatable system (49) is one of the best characterized and most widely used
`systems. It is based on the inhibitory action of the tet repressor (tetR) of Escherichia
`coli on the tet operator sequence (tetO). The tet-regulatable system most widely
`used in mammalian cells carries two modifications: The tetR is fused to the carboxy
`terminus of VP16 (a herpes virus transactivator), and the tetO-repeats are fused
`to a minimal human CMV promoter (Figure 3E). In the presence of tet, the tetR-
`VP16 fusion protein cannot bind to and activate tetO (tet-off system), whereas in
`the absence of tet, the tetR-VP16 protein can bind to tetO, resulting in increased
`expression levels of the gene of interest. An elegant way to deliver both the tet-
`off system and the gene of interest to the target cell is to use a single retroviral
`vector that contains both elements (66). The regulated expression of both the gene
`of interest as well as the regulatory sytem can be achieved by using an internal
`ribosomal entry site (IRES), resulting in bicistronic expression (Figure 3E). A
`drawback of this approach is that the tetR-VP16 fusion protein is toxic to cells.
`However, this problem can be overcome by placing tetR-VP16 under the control
`of the tetO-containing promoter (145). Also, the reverse tet-regulated system (50)
`in which the addition of tet induces transactivation (tet-on) has been successfully
`used in the context of retroviral vectors (90).
`Another family of chimeric-regulated systems is based on steroid hormones
`and their nuclear receptors. The organisms from which the hormones and their
`receptors have been isolated range from insects (ecdysone hormone of Drosophila
`melanogaster and Bombyx mori) to mammals (e.g., progesterone). The proges-
`terone system is based on a mutated human progesterone receptor (164). The
`binding domain of this receptor is fused to the yeast GAL4 DNA binding do-
`main and the VP16 domain, and it is activated by the antiprogesterone mifepri-
`stone (RU486), but not by the endogenous molecule present in mammals. In the
`presence of mifepristone, this chimeric regulator binds to the target gene, which
`contains the 17-mer GAL4 binding site, and activates transcription of the trans-
`gene (164). The use of the insect ecdysone-responsive system (117, 151) has the
`potential advantage that the ecdysone hormones are neither toxic nor known to af-
`fect mammalian physiology. In the Drosophila and the Bombyx-derived ecdysone-
`responsive system (DmEcR and BmEcR, respectively), the insect ecdysone recep-
`tor forms a heterodimer with the mammalian retinoid X receptor (RXR) (117).
`However, the DmEcR yields high levels of transactivation only if supraphysi-
`ological levels of RXR are present (117, 151). In contrast, the Bombyx-derived
`ecdysone-responsive system (BmEcR), presumably due to a higher affinity for
`RXR than the DmEcR, is capable of full transactivation with no added exogenous
`
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`RXR (151). Both ecdysone systems function in the context of retroviral vectors
`(151).
`Recently, a rapamycin-regulated transcriptional system was described for retro-
`viral vectors (128). Rapamycin mediates the formation of heterodimers between
`the immunophilin FK506-binding protein (FKBP) and the lipid kinase homolog
`FRAP (129). Fusion of the FKBP domains to a DNA-binding domain (called
`ZFHD1) and the rapamycin-binding domain of FRAP to a transcriptional activa-
`tion domain (derived from the p65 subunit of human NF-•B) forms a functional
`transcription factor that can be activated through rapamycin-dependent dimeriza-
`tion (135). This dimerizer-responsive transcription factor cassette can be incorpo-
`rated with the regulatable transgene into a single retroviral vector, which results
`in low basal expression levels and high dose-dependent induction of transgene
`expression. The induction ratios are in the range of three orders of magnitude and
`are comparable to the tet system.
`Retroviral transgene expression can also be controlled at the level of translation
`by inclusion of cis-acting posttranscriptional regulatory elements (PREs). PREs
`are present in herpes simplex, hepatitis B virus, and the woodchuck hepatitis virus.
`0
`of the
`The latter increases reporter gene expression at least fivefold if placed 3
`transgene in MLV-derived vectors (184).
`
`PACKAGING OF RETROVIRAL VECTORS To package the replication-defective vector
`into virions, the necessary viral proteins are provided in trans in the packaging cell
`(Figures 1 and 3). Retroviral packaging contructs are either transfected transiently
`into the packaging cells or a cell line [for a review over the presently available
`packaging cell lines, see (100)] is established that stably expresses the viral pro-
`teins. In either case, the packaging constructs are modified to reduce the chances
`of generating replication-competent virus (RCV) through recombination in the
`packaging cells. The cis-acting sequences required for packaging of the RNA (9),
`0
`LTR were deleted in the packaging constructs
`the polypurine tract, and the 3
`0
`0
`end of the 5
`LTR that contains the cis-acting sig-
`(93, 101). Furthermore, the 5
`nal sequence required for integration [the att element; for details see (30)] was
`0
`LTR with the CMV promoter resulted in a CMV-
`removed. Replacement of the 5
`driven packaging system that is compatible with the CMV/LTR hybrid vectors
`(see above) and results in high-titer virus preparations, especially if 293 cells are
`used (42, 116).
`To further minimize the extent of homology between the packaging constructs
`and the retroviral vectors, which could lead to the production of helper virus after
`a single recombination event in the packaging cells, a split genome packaging
`strategy was developed. In this case, two packaging constructs, one containing
`gag and pol and the other carrying env, are used (34, 94) (Figure 3G). Splitting the
`packaging genome into multiple units not only increases the safety of retroviral
`vectors but also facilitates pseudotyping of retroviral vectors with the envelope of
`different viruses.
`
`Annu. Rev. Genom. Hum. Genet. 2001.2:177-211. Downloaded from www.annualreviews.org
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`187
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`PSEUDOTYPING OF RETROVIRUS VECTORS The tropsim of retroviruses is deter-
`mined by the envelope glycoprotein, which binds to the receptor on the target
`cells. Ecotropic MLV can only infect murine cells that express the receptor for
`Env (a sodium-independent cationic amino acid transporter) (3). Fortunately, the
`host range of the vectors can be easily expanded by replacing the MLV env gene
`with envelope sequences from other retroviruses, e.g., amphotropic viruses that
`are able to infect mouse and nonmurine species (31, 102).
`Pseudotyping of retroviral vectors is not restricted to the envelope glycoproteins
`of other retroviruses. The G protein of the vescular stomatitis virus (VSV-G), a
`member of the rhabdovirus family, can substitute for the viral Env protein (21). The
`two major advantages of incorporation of the VSV-G protein are (a) the extremely
`broad host range of VSV, which enters the host cell by membrane fusion via the
`interaction with phospholipid components of the cell membrane (95) and (b) the
`ability to concentrate VSV-G pseudotyped particles more than 1000-fold (titers >
`109 IU/ml) by ultracentrifugation (21), which has important practical implications.
`The major disadvantage of VSV-G is that it is toxic to the packaging cells (21).
`Therefore, stable cell lines with inducible (e.g., tet-off system, see Regulation of
`Transgene Expression) VSV-G expression systems (26, 120) are required. On the
`other hand, transient transfection of the VSV-G expression plasmid, together with
`packaging constructs, circumvents this problem because harvesting is restricted to
`a relatively short period of several days following transfection.
`
`Lentiviral Vectors
`Lentiviruses are complex retroviruses, which have been named (lente, Latin for
`slow) according to the prototypic slowly progressing neurologic disease in sheep
`caused by the maedi/visna virus (70). An important genetic difference between
`simple retroviruses and lentiviruses are regulatory (tat and rev) and auxillary genes
`(vpr, vif, vpu, and nef ) that have important functions during the viral life cycle and
`viral pathogenesis (for details see 30, 64, 70). An outstanding feature of lentiviruses
`is their ability to infect nondividing, terminally differentiated mammalian cells,
`including lymphocytes and macrophages. This feature of lentiviruses makes them
`a very attractive tool for gene delivery (115, 158).
`
`HIV-BASEDVECTORS The first lentiviral vectors were derived from HIV-1 (114, 124,
`125, 134), the most extensively studied lentivirus. The HIV vector and packaging
`system are constantly evolving and serve as templates for the other lentiviral vec-
`tors. Apart from HIV-1, lentivirus vectors have been derived from HIV-2 (126),
`feline immunodeficiency virus (FIV) (127), equine infectious anemia virus (119),
`simian immunodeficiency virus (SIV) (92), and maedi/visna virus (12). Most of
`the lentiviral vectors presently in use for gene therapy approaches are HIV-derived
`vectors; therefore, we focus on these vectors. Similar to simple retroviruses, the
`cis- and trans-acting factors of lentiviruses can be separated while preserving
`
`Annu. Rev. Genom. Hum. Genet. 2001.2:177-211. Downloaded from www.annualreviews.org
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`PFEIFER ¥ VERMA
`
`their functions. The lentiviral packaging systems provide in trans the viral pro-
`teins that are required for the assembly of viral particles in the packaging cells.
`The vector constructs contain the viral cis elements, packaging sequences (9), the
`Rev response element (RRE), and th