1. Introduction
The basic principle of current gene therapy is to deliver genetic material to a population of cells in the body, thereby preventing a disease or improving the clinical status of a patient. One of key factors for successful gene therapy is the development of effective delivery. To date, a plethora of gene delivery systems, termed “vectors”, have been developed, and these fall into two broad categories: nonviral and viral vectors. Basically, the nonviral vector systems involve delivery of naked DNA or RNA into target cells with the aid of physical or chemical mediators such as cationic lipids. In terms of their simplicity, producibility, and immunogenicity, nonviral vector systems hold advantages over viral vector systems. However, in terms of the efficiency of gene delivery and expression, the viral vector systems are considered as more ideal (Goverdhana et al., 2005; Verma & Weitzman, 2005).
Although a variety of gene-transfer vectors based on RNA and DNA viruses have been adapted to deliver foreign genes to target cells
In contrast with adenoviral vectors, retroviruses have a substantial advantage as vectors for the sustained expression of a transgene in target cells (Verma & Weitzman, 2005). Retroviruses are enveloped RNA viruses belonging to the
It is well known that HIV-1 is the causative agent for acquired immunodeficiency syndrome (AIDS). Currently, the standard AIDS treatment, termed highly active antiretroviral therapy (HAART), is to use cocktail of antiretroviral drugs that targets different HIV-1 enzymes including reverse transcriptase and protease. However, although it successfully causes suppression of HIV-1 RNA detected in plasma for a prolonged period of time and dramatic decrease of patient mortality (Palella et al., 1998; Volberding & Deeks, 2010), this pharmacological therapy is facing problems such as drug resistance and side effects in administrated individuals (Meadows & Gervay-Hague, 2006; Richman, 2001). Hence, viral vector-based gene therapy should offer a new approach to supplement the need for current drug regimens for the treatment of HIV/AIDS (Poluri et al., 2003; Strayer et al., 2005). Genetic modification of HIV-susceptible cells or hematopoietic stem cells (HSCs) by expressing anti-HIV transgenes should be one of the goals of the HIV gene therapy (Kitchen et al., 2011). In this regard, the lentiviral vector has the potential advantage for transduction because of its ability to infect quiescent cells including HSC (Miyoshi et al., 1999). However, incorporation of anti-HIV genes into an HIV-based lentiviral vector can create problems for production of the vector itself; expression of the anti-HIV trans gene in the producer cells can interfere with vector production (Banerjea et al., 2003; Li et al., 2003; Mautino & Morgan, 2002c). One way to avoid this difficulty is to introduce a gene regulatable system in which the target transgene is kept silent during vector production and expression is subsequently induced on following infection of the vector in target cells.
Several regulatable gene expression systems have been developed and applied to viral vectors including lentiviral vectors (Goverdhana et al., 2005; Weber & Fussenegger, 2004). In this chapter, we focus on recent development of the gene-regulatable lentiviral vectors and discuss the suitability of the vectors for anti-HIV therapy.
2. Biology of HIV-1 replication
2.1. Genomic organization and gene expression
Lentiviruses, as represented by HIV-1, are also called complex retroviruses, which are characterized by a set of additional regulatory and accessory genes encoded in the viral genome (Cullen, 1991; Frankel & Young, 1998). In the case of HIV-1, the DNA genome converted from the RNA genome is about 9.7 kb and contains nine ORFs; in addition to the
The complexity of HIV-1 is also characterized by its specific pattern of viral gene regulation (Kingsman & Kingsman, 1996). The HIV-1 LTR harbors several
More than 30 species of RNA are transcribed from the integrated HIV-1 DNA; these fall into three size classes of mRNA based on the pattern of splicing: unspliced, partially spliced, and multiply spliced RNAs. (Kingsman & Kingsman, 1996; Purcell & Martin, 1993; Schwartz et al., 1990). The unspliced transcript is full-length RNA (about 9 kb) that is packaged as the viral genome into new viral particles but it also functions as mRNA to produce Gag and Gag-Pol polyproteins. The partially spliced transcripts (about 4 kb) encode Vif, Vpr, Vpu, and Env proteins. At early times after infection, however, the multiply spliced RNAs are predominant, and their encoded proteins, Tat, Rev, and Nef are highly produced (Kim et al., 1989).
2.2. Replication cycle
HIV-1 infection begins with the binding of the viral envelope (Env) glycoprotein gp120 (surface envelope protein: SU) to the CD4 receptor molecule on the surface of host cells. Consequently, the main target cells for HIV-1 infection are the CD4+ subset of helper T cells and monocyte/macrophage lineages (Dalgleish et al., 1984; Landau et al., 1988; Stevenson, 2003). The gp120-CD4 interaction triggers a conformational change in the gp120 that facilitates subsequent binding to a secondary cellular receptor (coreceptor). While most HIV-1 strains use either the α-chemokine receptor CXCR4 or the β-chemokine receptor CCR5 as the coreceptor, other chemokine receptors or related proteins have been reported to serve as coreceptors for HIV-1 infection (Berger et al., 1999). This coreceptor usage is the basis for the differential cell-type tropism of HIV-1 strains. Formation of the gp120-CD4-coreceptor complex then induces refolding of the gp41 subunit of the Env (transmembrane envelope protein: TM), which allows the membrane fusion process between the virus and target cell (Melikyan, 2008).
After penetrating the cell membrane, the viral nucleoprotein core, which contains genomic RNA, is released into the cytoplasm, followed by the uncoating of the viral core that is required for the formation of the reverse transcription complex (RTC) (Arhel, 2010; Bukrinskaya et al., 1998; Fassati & Goff, 2001). Reverse transcription, one of the defining steps of retrovirus infection, takes place in the RTC and it is initiated from the 3’ end of the tRNALys3 that is annealed to the primer binding site (PBS) near the 5’ end of the viral RNA genome. During the reverse transcription reaction, RT firstly synthesizes the minus-strand DNA along with the concomitant degradation of the RNA template by its RNase H activity (Basu et al., 2008). Subsequent synthesis of plus-strand DNA involves priming from two polyprine tracts (PPT), short RNA segments resistant to RNase H digestion, at the 3’ terminus (3’ PPT) and the center (central PPT: cPPT) of the HIV-1 genome. Once the 3’ end of the plus-strand DNA reaches the 5’ end of the cPPT, DNA synthesis proceeds by displacing the existing DNA fragments and stops at a central termination sequence (CTS) in the minus-strand DNA, resulting in a 99 bp triple-strand DNA structure in the center of the HIV-1 DNA (Arhel, 2010; Charneau et al., 1992; Charneau et al., 1994).
The newly synthesized full-length viral DNA remains associated with viral and cellular proteins in a large nucleoprotein complex called the preintegration complex (PIC) (Engelman, 2003). The HIV-1 PIC has been shown to contain RT, IN, matrix (MA), nucleocapsid (NC), and Vpr proteins (Lewinski & Bushman, 2005; Suzuki & Craigie, 2007). In addition to viral proteins, several cellular proteins have been reported as components of the HIV PIC (Suzuki & Craigie, 2007). As mentioned above, unlike many oncoretroviruses, HIV-1 is able to infect non-dividing cells. Thus, the HIV-1 PIC is believed to carry karyophilic signals that direct transport across an intact nuclear membrane in non-dividing cells. Although the molecular mechanisms underlying the active transport of HIV-1 PIC into the nucleus is still poorly understood, several viral and cellular factors have been shown to be implicated in the nuclear import of the HIV-1 PIC (Fassati, 2006; Suzuki & Craigie, 2007; Yamashita & Emerman, 2006). Cell cycle-independent infection of HIV-1 is particularly important in the pathogenesis of the virus and the development of HIV-1-based lentiviral vectors (Blankson et al., 2002; Kaul et al., 2001; Somia & Verma, 2000).
Following nuclear transport of the PIC, integration of viral DNA into the host chromatin takes place. IN is a key component of the PIC that catalyzes the integration. This reaction proceeds via three coordinated steps: 3’ end processing of the viral DNA, joining to the target DNA, and repairing of the gaps between viral DNA and target DNA. IN is responsible for the 3’ end processing and DNA joining steps, but the gap repair step is likely to be carried out by yet-to-be-identified cellular enzymes (Engelman, 2003) (Fig. 2).
The integrated DNA, called the provirus, is acted upon by cellular transcription factors to express viral genes. Early populations of the transcripts are the multiply spliced class of mRNA that encodes Tat, Rev, and Nef proteins (Kingsman & Kingsman, 1996). Tat enhances production of viral mRNAs by more than two log via interaction with TAR and a cellular elongation complex (Brady & Kashanchi, 2005). There is then an increase in the partially spiced and unspliced mRNAs along with a concomitant decrease in the multiply spliced mRNAs, which is caused by the accumulation of Rev protein. Rev is also required for the nuclear export of partially spliced and unspliced mRNAs. These classes of viral RNAs contain a highly structured
Following the synthesis of the full-length viral RNA genome and the viral proteins, these components are assembled together to produce new viruses. In HIV-1, the assembly process takes place at the plasma membrane (Ono, 2010). Gag protein plays a central role in the formation of virions; this protein is synthesized as a 55 kDa precursor protein for matrix (MA), capsid (CA)), and nucleocapsid (NC) proteins. The Gag precursor proteins are rapidly targeted to the plasma membrane where they multimerize. Although the multimerization of the Gag is sufficient to give rise to virus like particles, incorporation of Gag-Pol proteins is integral to the formation of infectious virions (Wu et al., 1997). Gag-Pol is a 160 kDa multidomain protein consisting of RT, IN, and protease (PR), and it too relocates to the plasma membrane, where Gag and Gag-Pol are assembled into virus particles. In the HIV-1 genome, Gag and Pol are encoded by overlapping ORF; Gag-Pol is generated by a ribosomal frameshifting during translation of the
Accessory proteins (i.e. Vif, Vpr, Vpu, and Nef) are dispensable for viral replication in many
3. Development of HIV-1-derived lentiviral vectors
Although retrovirus vectors derived from oncoretroviruses were introduced first, in recent years, attention of the viral vector research has been focused on lentiviruses such as HIV-1 and equine infectious anemia virus (EIAV) due to their ability to infect non-dividing cells. In particular, HIV-1 should be one of the most practical gene transfer vectors for gene therapy applications because this is the best studied retrovirus. However, HIV-1 is a human pathogen that causes destruction the of CD4+ helper T lymphocytes and the subsequent loss of immune competence (Forsman & Weiss, 2008). Therefore, considerable efforts have been devoted to develop efficient HIV-1-derived lentiviral vectors with improved biosafety features.
So far, three different generations of HIV-1-based lentiviral vectors have been established, which is based on the level of safety improvements in viral vector production (Fig. 3).
3.1. First-generation lentiviral vectors
One of the key safety concerns in the use of HIV-derived vectors is the generation of replication competent lentiviruses (RCL). Earlier development of lentiviral vectors was achieved by transient transfection of human embryonic kidney (HEK293T) cells with three separate plasmid DNAs encoding i) the lentiviral vector genome which was composed of the wild-type 5’ and 3’ LTR, a part of the
3.2. Second-generation lentiviral vectors
The second-generation lentiviral vectors basically employ a similar three-plasmid system as the first generation vectors. Yet, in order to overcome the safety issue attributable to the first-generation vectors, the second-generation lentiviral vectors were generated without production of all accessory proteins (Vif, Vpr, Vpu, and Nef) via mutation or deletion of these genes from the packaging plasmid (Gasmi et al., 1999; Kim et al., 1998; Zufferey et al., 1997) (Fig. 3B).
3.3. Third-generation lentiviral vectors
The second-generation vectors, however, still carry the transcriptionally active LTR elements that could induce the homologous recombination between the vector genome and wild-type HIV-1. This would be particularly problematic if the lentiviral vectors are used for gene therapy of HIV/AIDS. Thus, further improvements were made in the third-generation lentiviral vectors. To minimize the transcriptional activity of the LTR in transduced cells, the enhancer/promoter unit was deleted from the U3 region of the 3’ LTR in transfer vector plasmids. During reverse transcription in the transduced cells, this deletion is transferred to the 5’ LTR of the lentiviral DNA, thereby reducing promoter activity of the integrated provirus (self-inactivating [SIN] vector) (Miyoshi et al., 1998; Zufferey et al., 1998). Additionally, the U3’region of the 5’ LTR in the transfer vector plasmid was replaced with the cytomegalovirus (CMV) promoter, which enabled Tat-independent transcription of the lentiviral vector genome in producer cells (Dull et al., 1998; Miyoshi et al., 1998). In these SIN vectors, there is no compete HIV-1 U3 sequence. Moreover, expression of Rev protein is directed by a separate plasmid, but not by the packaging plasmid encoding
4. Incorporation of regulatable gene expression systems in HIV-1-derived lentiviral vectors
Lentiviral vectors hold great promise for a gene therapy approach to inherited and acquired diseases. In these particular clinical settings, it would be more beneficial to reversibly control transgene expression in a dose and time dependent manner as illustrated in the field of angiogenesis and Parkinson’s disease (Ma et al., 2002; Yancopoulos et al., 2000). To meet the standards required for clinical applications, a number of regulatable gene expression systems have been developed, and some of them are indeed incorporated into viral vectors including HIV-1-derived lentiviral vectors (reviewed in Goverdhana et al., 2005).
Although early development of the regulatable gene expression systems was based on naturally occurring inducible cellular promoters that respond to exogenous signals, these types of systems had limitations due to the pleiotropic effects of the inducer, high levels of “leaky” background expression and poor performance in inducibility. Therefore, recent efforts have been mostly focused on the development of chimeric gene regulatable systems derived from prokaryotic, eukaryotic, and viral elements, which are designed to enhance specificity and activity of transgene expression (Fussenegger, 2001).
4.1. Tetracycline-regulated system
The most widely used inducible system in lentiviral vectors is the tetracycline (Tet)-regulated system. This system was originally based on binding of the Tet-controlled repressor (tetR), a 23.6 kDa protein of
In addition to the background activity in the repressed state, the major limitation of the Tet-off system is the requirement of continuous administration of Tet or Dox to suppress transgene expression. To overcome this limitation, another type of Tet-controlled gene expression system was established by introduction of several permutations in the tTA protein of the Tet-off system. The mutant tTA binds the tetO sequences only in the presence of Tet/Dox: it exhibits opposite function (Gossen et al., 1995). Because this modified version of regulatable system, the so-called Tet-on system, shows rapid kinetics of gene upregulation compared to Tet-off system, several HIV-1-derived lentiviral vectors have been constructed using the Tet-on system as well (Johansen et al., 2002; Koponen et al., 2003; Pluta et al., 2005; Reiser et al., 2000; Vogel et al., 2004) (Fig. 4B).
One more approach to permit tight control of transgenes in the context of a lentiviral vector is demonstrated in the Tet-regulated system that employs a chimeric tetR fused with the Krüppel-associated box (KRAB) domain, a transcriptional regulator found in many DNA binding zinc-finger proteins (Szulc et al., 2006; Wiznerowicz & Trono, 2003). Binding of the KRAB domain-containing repressor protein to DNA recruits various heterochromatin-inducing factors, thereby suppressing activity of cellular RNA polymerases (RNAPs) II and III. This transcriptional silencing can be exerted no farther than 2-3 kb away from the repressor binding site. In the new design of Tet-regulated lentiviral vector system, the tetO site was inserted upstream of the RNAP III promoter-driven small hairpin RNA (shRNA) expression cassette which was located in the U3 region of lentiviral vector genome, and the activity of tetO-linked shRNA expression unit was tightly suppressed in the presence of KRAB-fused tTA and in the absence of Dox. The KRAB-fused tTA/Dox-dependent inhibition of transcriptional activity was also observed in the internal RNAP II promoter for a reporter transgene within the same vector genome. However, when the transduced cells were treated with Dox, shRNA was produced to achieve RNA interference, which was correlated with the expression of the reporter transgene (Szulc et al., 2006; Wiznerowicz & Trono, 2003).
One drawback of the Tet-regulated system is that there is a requirement to deliver two distinct expression units into a target cell: one is for transactivator (e.g. tTA) expression and the other is for transgene expression. In a binary lentiviral vector approach in which tTA and transgene expression cassettes are cloned into separate vectors, a population cells that is singly transduced with either tTA or transgene would be produced, resulting in low inducibility as a whole. This can be a bottleneck, particularly in relevant applications of the Tet-regulated lentiviral vector systems in clinical use. Single vector systems that contain the entire regulatable component in a unique vector is one of the solutions to guarantee simultaneous expression of the two expression units in the target gene, and these have indeed been established (Gascon et al., 2008; Kafri et al., 2000; Szulc et al., 2006; Vogel et al., 2004).
4.2. Mifepristone-inducible system
This gene regulatable system (also called GeneSwitch system) is based on a mutated human progesterone receptor that responds to the synthetic progestin antagonist but fails to bind natural progestins or other steroids (Burcin et al., 1999; Wang et al., 1994). Similar to Tet-regulated system, this system requires two components: the regulator (transactivator) protein and the inducible promoter sequence that drives transgene expression. The regulator is a hybrid protein consisting of a GAL4 DNA-binding domain from
Sirin and Park have incorporated the MFP-inducible gene expression system into HIV-1-derived lentiviral vectors (Sirin & Park, 2003). In their design, two different SIN lentiviral vectors were constructed, in which either the regulator protein expression unit or the inducible transgene expression unit was cloned. When human cell lines (HeLa and Huh7 cells) were infected with both lentiviral vectors, up to a 275-fold increase in the number of reporter fluorescent protein-positive cells was observed at 48 hours following MFP treatment. Similar effective induction was also detected in cells transduced by a lentiviral vector expressing the human α1-antitrypsin (hAAT) with an extremely low level of basal hAAT expression (Sirin & Park, 2003).
Although an
4.3. Ecdysone-regulated system
The
In the single vector approach, the inducible GFP expression cassette containing E/GRE as well as a CNV promoter-driven bicistronic unit for VgEcR and RXR expressions were cloned into an HIV-1-derived SIN lentiviral vector. However, this ecdysone-regulated system was also applicable to the binary vector system, in which the VgEcR/RXR expression unit was in one lentiviral vector and the inducible transgene expression unit was in the second vector. The latter approach would be of value in deliver of longer transgene. These lentiviral vectors have been shown to successfully deliver the ponA-inducible GFP expression units
4.4. Other regulatable systems
Besides the Tet- MFP-, and ecdysone-regulated systems, different types of inducible lentiviral vectors have been generated based on the other chimeric regulatable systems, which include streptogramin-adjustable expression system derived from
5. Application of a gene regulatable lentiviral vector for HIV-1 inhibition
Lentiviral vector-mediated gene therapy has the potential to improve the clinical state of a patient with HIV-1. This goal would be accomplished by
Although a promising approach for HIV gene therapy, constitutive expression of anti-HIV genes in the context of HIV-1-derived lentiviral vectors could encounter a problem of self-inhibition of the vector particle production, resulting in significant decrease of viral infectious titer (Mautino & Morgan, 2002b). This problem of self-inhibition can be solved by several means. If the anti-HIV gene targets specific sequences in the HIV-1 RNA, one strategy to avoid the self-inhibition would be to engineer nucleotide sequences of lentiviral vector genome and packaging genes in order that anti-HIV gene will exclusively recognize wild-type virus in transduced cells. As for TdRev, the inhibition of vector production could be overcome by replacement of the HIV-1 Rev-dependent nuclear export element (e.g. RRE) with the one derived from another lentiviruses, conferring Rev-independent property on the lentiviral vectors (Mautino et al., 2001; Taylor et al., 2008). But, if the anti-HIV genes are designed to target HIV-1 at more fundamental process such as virion formation and budding processes, an ideal strategy would be the incorporation of regulatable transgene expression system into lentiviral vectors, in which expression of the anti-HIV gene is “OFF” during vector production and turned “ON” in the target cells.
In order to assess the availability of gene regulatable systems in inhibition of HIV-1, we have generated HIV-1-derived lentiviral vectors harboring the MFP-inducible transgene expression unit (Shinoda et al., 2009). In the study, two SIN lentiviral vectors were designed to incorporate the MFP-inducible unit in either the forward or the reverse orientation with respect to the direction of transfer vector genome (designated as forward and reverse vectors, respectively), since it has been reported that promoter activity of the internal gene expression unit could be affected by its orientation and/or the presence of adjacent LTR (Chen et al., 1992; Reiser et al., 2000; Sirin & Park, 2003). When firefly luciferase gene, which does not interfere with HIV-1 production, was inserted into the MFP-inducible lentiviral vectors, substantial levels of infectious vectors could be yielded from the forward and reverse vector systems by co-transfection with packaging plasmid DNAs in HEK293T cells. However, the infectious titer obtained by the forward vector was more than 10-fold higher than reverse vector, and even in the absence of transactivator and inducer, significant level of the leaky expression of luciferase was observed in the forward vector plasmid transfected-cells, but not in the reverse vector-transfected cells (Shinoda et al., 2009). It can be speculated that the higher background activity in the forward vector was due to the enhancement of gene expression by orientation-dependent
6. Conclusion and future direction
Lentiviral vectors derived from HIV-1 are attractive gene delivery vehicles in terms of stable and long-term transgene expression in dividing and non-dividing cells. Although strong promoters used to achieve high levels of transgene expression are put into general use in the lentiviral vectors, incorporation of regulatable gene expression system confers transcriptional flexibility to the vector, which expands the potential of the lentiviral vectors for a wide array of gene transfer applications, particularly when undesired side effect would be expected by the constitutive expression of transgene. Nevertheless, many obstacles must be overcome for the clinical application of gene regulatable lentiviral vectors in gene therapy. One of the obstacles is that all the components of a regulatory system should be incorporated into a single vector, limiting the cloning capacity for transgene. Another issue is that there is no gene regulatory system approved by the FDA for clinical use. However, if their safety and efficacy are validated, development of gene regulatable lentiviral vector systems will be a next promising step toward achieving successful gene therapy for otherwise incurable diseases.
Acknowledgments
We are grateful Wei Xin Chin for proofreading and comments on the manuscript.
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