Lentiviral Vectors: Their Molecular Design,Safety,and Use in Laboratory and Preclinical Research

Abstract

Lentiviral vectors have been successfully used in the clinic and they are increasingly being used for nonclinical applications. They are capable of stably transducing a broad range of mammalian cell types, including nondividing cells, with high efficiency. This review summarizes the evolving molecular design of lentiviral vectors, describing how they have improved since their first description. Lentiviral vector safety and issues surrounding genotoxicity are discussed. Examples of successful application of lentiviral vectors in laboratory and preclinical research are described. These include functional genomics, target validation, protein manufacturing, in vivo imaging, transgenic animals, and stem cell research.

Introduction

I n the biological sciences, vectors are vehicles used for the delivery of DNA or RNA into cells. Because nucleic acids do not efficiently enter cells unaided, agents such as calcium phosphate, lipid, electric current, and viral vectors are necessary for satisfactory transfer of DNA into the nucleus for gene expression. Although many of these agents are adequate, viral vectors are the most proficient at gene transfer, because the viruses from which they are derived are endowed with sophisticated machinery that facilitates efficient cell entry, transport, and expression of their genomes in the nucleus of target cells. Viral vectors derived from the Retroviridae family of viruses are of special interest because they convert their RNA genomes into DNA and integrate them into the chromosomes of target cells, a mechanism facilitated by reverse transcriptase and integrase (Baltimore et al., 1974; Esposito and Craigie, 1999). The integrating property of retroviruses is what makes them useful for research. Daughter cells derived from retroviral vector-transduced cells are not cured of the transferred genomes, but maintain them in their chromosomes after cell division. This stable transduction allows for the creation of cell lines with specific genetic modifications that are useful for research and other purposes.

Lentiviruses are distinct members of the Retroviridae family of viruses. Lentiviral vectors (LVs) have been constructed from several types of lentiviruses, but the most commonly used is the human immunodeficiency virus or HIV, a virus whose molecular biology has been extensively studied (Rabson and Martin, 1985; Joshi and Joshi, 1996; Nielsen et al., 2005; Pluta and Kacprzak, 2009). Lentiviral vectors have the unique advantage of being able to stably transduce dividing and nondividing cells (Naldini et al., 1996; Naldini, 1998). In contrast to oncoretroviral preintegration complexes (PICs), the lentiviral PIC has the capability of active transport into the nucleus through nuclear pore complexes in an ATP-dependent manner (Bukrinsky, 2004), essential for efficient transduction of nondividing cells. What makes lentiviral vectors distinctly useful is their ability to stably transduce a wide variety of mammalian cell lines and primary cells with high efficiency (Davis et al., 2004; Lu et al., 2004; Salmon and Trono, 2007).

Although lentiviral vectors have been safely used in human clinical trials (Levine et al., 2006; Cartier et al., 2009; Cavazzana-Calvo et al., 2010), the focus of this review is on their use in laboratory and preclinical research. Nevertheless, safety and other data garnered from clinical use of lentiviral vectors provide increasing confidence for their continued and expanded laboratory use. This review addresses the iteratively improving molecular design and safety of lentiviral vectors, providing examples of their application in the laboratory and preclinical research setting.

The Evolving Molecular Design of Lentiviral Vectors and Their Safety

Lentiviral vectors have been developed with HIV-1 (Dull et al., 1998), HIV-2 (D'Costa et al., 2001), feline immunodeficiency virus (FIV) (Poeschla et al., 1998), equine infectious anemia virus (EIAV) (Mitrophanous et al., 1999) and simian immunodeficiency virus (SIV) (Mangeot et al., 2000). There has been no study that evaluates the comparative safety of one class of lentiviral vector over another, although investigators have their preferences and supporting arguments. One line of thought is that non-HIV vectors are safer because they are derived from viruses that are not known to infect humans (Gilbert and Wong-Staal, 2001). The contrary argument proposes that it is safer to develop vectors from a virus whose molecular biology and pathogenesis are well understood and for which multiple drug therapies are available, than from viruses whose pathogenicity in humans is uncertain (Dropulić, 2001). Although non-HIV LVs have certainly been used, the overwhelming majority of laboratory and clinical studies have been performed with HIV-1-based LVs in human cells.

HIV-1-based lentiviral vectors were originally derived from molecularly cloned proviruses that were developed in the 1980s (Adachi et al., 1986; Ratner et al., 1987). They have undergone iterative improvement since their first description (Poznansky et al., 1991; Parolin et al., 1994; Naldini et al., 1996). The improvements have largely focused on the vector and helper design, which are constructed as plasmid DNAs that are transfected into HEK 293 or 293T cells to produce viral vector particles. It is worth noting that the first LVs were not pseudotyped with a heterologous envelope protein, but with the native HIV gp160/120 envelope protein. This limited their tropism to CD4-expressing cells, the natural target of HIV. The broad potential of HIV-1-based lentiviral vectors was realized on two discoveries: that they efficiently transduce nondividing cells and that retroviral particles can be pseudotyped with heterologous envelope proteins such as vesicular stomatitis virus G envelope protein (VSV-G), to confer broad tropism for transduction of a wide variety of mammalian cell types (Emi et al., 1991; Bukrinsky et al., 1993; Yee et al., 1994; Naldini et al., 1996; Naldini, 1998).

Vector and Helper Configurations

HIV-1-based lentiviral vectors include a transducing vector and separate helper (packaging) plasmids. Although not formally proven, it is believed that the separation of such components decreases the likelihood of recombination and therefore the theoretical generation of a replication-competent lentivirus (RCL), which could be potentially harmful to humans. It is important to note that no such RCL has ever been observed despite large-scale production and testing of lentiviral vectors (Manilla et al., 2005; Cornetta et al., 2011).

Helper components consist of structural and enzymatic proteins (Gag and Pol), which are required for virion formation. The first helper constructs were simply deleted in HIV-1 envelope (Env), with the VSV-G envelope provided in trans on a separate plasmid. However, this has not always the case; the first lentiviral vector safely tested in human clinical trials contained all helper components on a single plasmid, where the Gag-Pol open reading frame (ORF) and VSV-G ORF were separated by a triple cis-acting ribozyme cassette to prevent transcriptional readthrough (Lu et al., 2004; Levine et al., 2006). Nevertheless, helper constructs have been iteratively designed to separate and remove HIV open reading frames to help ensure their safety. So-called second-generation helper constructs were developed to comprise the Gag and Pol open reading frames, which encode the structural and enzymatic proteins of the vector particle and the Tat and Rev genes, which upregulate transcriptional activity and export of genomic RNA to the cytoplasm, respectively. The accessory genes, known to be important for pathogenesis of the wild-type virus (Desrosiers et al., 1998), were deleted to help improve safety in case of recombination. The native long terminal repeat (LTR) promoter was substituted with a heterologous promoter such as the cytomegalovirus (CMV) promoter, obviating the need for both Tat enhancement of transcription and the requirement for helper Tat protein expression. It is known that Tat-deleted mutants of wild-type HIV are not replication competent (Vendel and Lumb, 2003; O'Brien et al., 1990). Therefore, the deletion of Tat could decrease the risk of generating a putative RCL.

The transfer vector is used to transfer the gene(s) of interest to target cells. These have also been iteratively improved to match the helper constructs. Early transfer vectors were composed of a 5′ LTR, major splice donor site, packaging signal encompassing the 5′ part of the Gag gene (in contrast to the first HIV-based vectors, which omitted the packaging signal components in Gag and therefore were low in titer; Poznansky et al., 1991), the Rev-responsive element (RRE), the envelope splice acceptor, an internal gene cassette driven by its own promoter, and the 3′ LTR. The 3′ LTR was modified to delete the U3 region, which is essential for replication of the wild-type virus. However, the U3 region of the 3′ LTR is dispensable for a replication-defective vector, because expression of the packaged genomic RNA is driven from the 5′ LTR and the transgene is expressed from a promoter located within the vector. Removal of enhancers and other transcriptionally active sequences from the retroviral 3′ LTR results in a self-inactivating (SIN) LTR (Yu et al., 1986). This is considered safer than native LTR-containing vectors, but formal proof of their purported increased safety is lacking (Buchholz and Cichutek, 2006).

Successive iterations of transfer vectors aimed to improve safety and transgene capacity by removing as much of the native viral sequences as possible. There is, of course, a limit to this, a point at which further deletion starts to impact vector titer. For example, removal of the RRE sequence and associated splice donor and acceptor sequences results in a loss of transduction efficiency (Kim et al., 1998). In fact, sometimes an improvement in vector design was achieved by adding sequences back. Adding approximately 100 nucleotides of the central polypurine tract region (central DNA flap) of HIV back to the vector resulted in more efficient reverse transcription and nuclear import of the vector genome, thereby increasing transduction efficiency (Dardalhon et al., 2001; Zennou et al., 2001; De et al., 2005). In other cases, heterologous elements have improved the properties of the transducing vector. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been widely used to stabilize transgene mRNA levels and therefore increase transgene expression (Zufferey et al., 1999; Dupuy et al., 2005). However, there have been concerns surrounding the potential oncogenic activity of the native WPRE, because of the expression of an oncogenic WHV-X protein from an open reading frame found within the element (Kingsman et al., 2005). The expression of the protein was abrogated simply by mutating the WHV-X ORF translation start site, which did not affect WPRE function, thereby removing the oncogenic safety risk (Zanta-Boussif et al., 2009). Other heterologous elements have been incorporated into lentiviral vectors, with mixed success. Insulator and similar elements held the promise of partitioning transgene expression from regions of the chromosome that surround the site of vector integration, and thereby reducing potential genotoxic effects of vector insertion. However, no conclusive proof has been shown that insulators robustly perform this function without adversely affecting vector titer or function (Hanawa et al., 2009; Uchida et al., 2011). In fact, in some cases insulators can be genotoxic themselves, significantly reducing their potential utility (Grandchamp et al., 2011).

Other elements have been more successfully incorporated into lentiviral vectors, some for specific purposes. Tetracycline (Tet) trans-activators or repressors have been used to create inducible expression lentiviral vectors (Pluta et al., 2007). Although early versions had issue with basal activity (Kafri et al., 2000), optimized versions avoided this limitation and demonstrated low basal and high transgene inducibility when combined with silencing elements such as KRAB (Krüppel-associated box) (Wiznerowicz and Trono, 2005; Bulliard et al., 2006; Laurenti et al., 2010). An alternative method for inducible gene or short hairpin RNA (shRNA) expression uses Cre–loxP recombination, in which the bacteriophage P1 Cre recombinase is used to remove DNA that is flanked by two loxP sites (Kuhn and Torres, 2002). Cre–loxP-inducible lentiviral vectors contain two loxP sites that are inserted between the promoter and the transgene or shRNA, with a stuffer sequence in between that hinders shRNA expression. On the addition of Cre, the stuffer between the loxP sites is removed and expression of the gene or shRNA is initiated. Unlike the tetracycline system, this mode of transcriptional activation is generally irreversible and has particular use in transgenic animals for the study of genes that would otherwise generate embryonic lethality (Pluck, 1996; Chang and Zaiss, 2003).

Lentiviral vectors continue to evolve in their molecular design. More recently, it has been found that the incorporation of cell type-specific microRNAs downstream of a transgene can restrict expression in those particular cell types (Brown et al., 2006, 2007). The combination of positively regulating tissue-specific promoters with negatively regulating microRNAs can result in highly precise transgene expression in specific cells or tissues (Brown and Naldini, 2009). This technology may be particularly useful as a mechanism to increase vector safety, by limiting transgene expression only to target cells. As the demand for experimentation in specific cell types increases, technologies that precisely regulate gene expression will become more important for research and clinical applications.

Lentiviral Vectors and Genotoxicity

The safety of oncoretroviral and lentiviral vectors has been examined since the unfortunate observation of leukemia in patients with X-linked severe combined immunodeficiency (SCID-X1) who were transplanted with hematopoietic stem cells modified with a murine oncoretroviral vector expressing the common γchain of the interleukin-2 receptor (IL-2R) gene (Hacein-Bey-Abina et al., 2003, 2008). Analysis of patients' cells showed that the murine oncoretroviral vector integrated in proximity to the LMO2 proto-oncogene promoter. This resulted in aberrant transcription and expression of the LMO2 gene and triggered unregulated cell proliferation that was driven by enhancer elements in the oncoretroviral vector LTR acting on the LMO2 gene promoter. A series of studies were initiated to better understand the genotoxic risk of oncoretroviral and lentiviral vectors. In an important study, Naldini and colleagues compared the relative genotoxic potential of oncoretroviral vectors and lentiviral vectors (Montini et al., 2006). Using the Cdkn2a^–/– mouse transplantation model, which is susceptible to a broad range of cancer-triggering genetic lesions, they found that oncoretroviral vectors triggered dose-dependent acceleration of tumor onset, whereas tumorigenesis was unaffected by lentiviral vectors, despite a higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. The results of these studies have been confirmed by others and indicate a favorable safety profile for lentiviral vectors (Maruggi et al., 2009; Modlich and Baum, 2009; Modlich et al., 2009; Montini et al., 2009). Current clinical experience with lentiviral vectors supports these experimental studies. There have been no oncogenic events observed in human clinical trials using lentiviral vectors to date (Levine et al., 2006; Cartier et al., 2009; Cavazzana-Calvo et al., 2010). However, clonal expansion and dominance of hematopoietic progenitors have been reported in a clinical trial in which hematopoietic stem cells were genetically modified with a lentiviral vector that expressed the β-globin gene for the treatment of thalassemia (Cavazzana-Calvo et al., 2010). Although the mechanisms for clonal dominance remain unclear, it appears that low transduction efficiencies may actually increase the probability for clonal events because of the potential polyclonal nature of the hematopoietic stem cell population (Cavazzana-Calvo et al., 2011; Maetzig et al., 2011). These findings suggest that vector design, purity, and transduction methods are important considerations when designing clinically relevant studies and for interpreting their data, especially in genotoxic sensitive cell types such as stem cells. It is worth noting that genotoxicity appears to affect primarily stem cells and not somatic cells. For example, T cells transduced with oncoretroviral vectors are highly resistant to oncogenic transformation (Newrzela et al., 2008; Cattoglio et al., 2010).

Useful guidelines have been developed by regulatory agencies for using lentiviral vectors in laboratory, preclinical, and clinical research. In the United States, some general criteria should be considered when conducting risk assessments for research involving lentiviral vectors, which are discussed in the document “Risk assessment and containment determination for research with lentiviral vectors,” published by the U.S. National Institutes of Health (NIH, Bethesda, MD) Recombinant DNA Advisory Committee (RAC) (this document can be found at http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdf).

Guidance has also been published in 2005 by the European Medicines Agency (EMEA, London, UK) Committee for Medical Products for Human Use (CHMP): “Guideline on development and manufacture of lentiviral vectors,” which can be found at www.emea.europa.eu/pdfs/human/bwp/245803en.pdf.

Functional Genomics Applications

Lentiviral vector-mediated gene expression and knockdown have been successfully used to understand gene function. Initially, individual genes were either overexpressed or knocked down with shRNA (Levy et al., 2002; Chilov et al., 2003; Li and Rossi, 2005). With the advent of the genomics era, a superior approach is to identify gene function on a genome-wide basis. Lentiviral vector libraries that either overexpress cDNA or shRNA that inhibit specific gene function have been reported (Kawano et al., 2004; Guryanova et al., 2006; Moffat et al., 2006; Root et al., 2006; Kurita et al., 2008; Shtutman et al., 2010). Nevertheless, single-target knockdown of gene expression continues to play an important role in the validation of drug targets. For instance, Benson and colleagues validated the action of two members of the polycomb group of transcriptional repressor proteins, Bmi-1 and Mel-18, in several cancer cell lines, using Tet-inducible LV shRNAs (Wiederschain et al., 2009). Knockdown of Bmi-1 and Mel-18 expression rapidly revealed tumor cell dependence on the expression of these proteins.

Several lentiviral vector cDNA libraries have been described (Chilov et al., 2003; Kurita et al., 2008). Koyanagi and colleagues (Kawano et al., 2004) found that when an LV cDNA library was transduced into CD4⁺ T cells and later challenged with wild-type HIV-1, a restricted set of cells acquired resistance to HIV-1-induced cytopathic effect (CPE). Analysis of the resistant LV-transduced cells revealed overexpression of the human CD14 gene. CD14 overexpression on CD4⁺ T cells was subsequently shown to inhibit the entry of HIV-1 and HIV-1-induced CPE. More recently, a fetal liver LV cDNA library consisting of more than 8 × 10⁷ clones has been developed (Kurita et al., 2008). Several commercial sources have reported the availability of various lentiviral vector cDNA libraries.

The RNAi Consortium at the Broad Institute (Cambridge, MA) pioneered the development of a loss-of-function screening platform using lentiviral vectors. The LV shRNA libraries contained more than 100,000 lentiviral vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs (Moffat et al., 2006; Root et al., 2006). By analyzing a restricted set of 5000 unique shRNA-expressing LVs that targeted 1028 human genes, approximately 100 candidate regulators of mitotic progression and proliferation were identified. A similar approach has been used to identify novel specific gene targets capable of altering hematopoietic stem cell expansion and differentiation (Ali et al., 2009). More recently, Roninson and colleagues have developed an inducible LV shRNA library, using the Tet–KRAB system (Shtutman et al., 2010). They generated a library consisting of 2.8 × 10⁶ clones, using a cDNA digestion and hairpin-adaptor ligation strategy. Sampling the library showed that 79.1% of the shRNAs matched with the UniGene transcript database (www.ncbi.nlm.nih.gov/unigene), and 12% of the clones matched human genome sequences but not sequence in the database and are likely to represent yet-unidentified transcripts. Massive parallel sequencing revealed a relatively unbiased library because 72% of the LV shRNA clones differed in representation by no more than 3-fold. The investigators were able to verify gene pathways responsible for breast carcinoma cell growth, using a selection strategy after transduction of MDA-MB-231 cells with the library. Of the 53,000 sequences analyzed by massive parallel sequencing, 9 of the 10 gene pathways were associated with various cancers, cell cycle progression, or apoptosis. Also, Haber and colleagues performed a genome-wide RNA interference (RNAi) screen, using 55,000 pooled lentiviral shRNAs targeting 11,000 genes, to define the functional pathways regulating epithelial cell migration (Smolen et al., 2010). They were able to identify a set of 31 genes representing diverse pathways whose knockdown dramatically enhanced cellular migration. They observed universal activation and a profound dependence on extracellular-signal regulated kinase (ERK) and ribosomal S6 kinase (RSK), and were able to validate this dependency pharmacologically. The study revealed a pharmacological opportunity to inhibit cell migration, including cancer metastases, via the pathways identified with the LV shRNA screen.

Cell-Engineering Applications

Lentiviral vectors are being increasingly used for the genetic modification of primary cells, which are then employed for various applications. In vivo imaging studies of cells have become increasingly important as investigators seek to understand cell migration and function in animal models. Verma and colleagues initially described in vivo imaging of hepatocytes transduced with the green fluorescent protein (GFP) in mice (Pfeifer et al., 2001). Imaging has been frequently used for ascertaining in vivo cell distribution and transgene expression in a variety of settings, including nonhuman primates (Sander et al., 2006; Tarantal et al., 2006; Lin et al., 2011; Warlich et al., 2011). Although several reporter genes have been used, a wild-type or mutant variant of herpes simplex virus type 1 (HSV1-tk-sr39) has been popular (De et al., 2003; Jang et al., 2010). To improve visualization of marked cells, Gambhir and colleagues (Ray et al., 2004) developed a lentiviral vector composed of a triple fusion reporter gene expressing a synthetic Renilla luciferase reporter gene, a reporter gene encoding the monomeric red fluorescence protein, and HSV1-tk-sr39. They showed that metastases of the human melanoma cell line (A375M) stably expressing the triple fusion could be imaged by micro-positron emission tomography (microPET) and optical technologies over a 40- to 50-day time period in living mice. An ever-expanding range of cell types is being examined by in vivo imaging. Of particular interest is the imaging of stem cells so as to understand their in vivo migration and differentiation properties. Lentiviral vectors expressing the firefly luciferase (fLuc) gene were used to monitor human embryonic stem cell (hESC) engraftment and proliferation in live mice after transplantation (Pomper et al., 2009). Transduction was shown not to alter the properties of hESCs in culture. Teratoma formation occurred from both gene-modified cells as well as wild-type hESCs 2–4 months after inoculation in mice. Using an optical imaging system, bioluminescence from the LV-fLuc-transduced hESCs was detected in mice bearing teratomas well before palpable tumors could be detected. However, expression of multiple reporters is not without its consequences. Microarray profiling of embryonic stem cells expressing the triple reporter revealed the downregulation of cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and antiapoptosis genes (Wu et al., 2006). Although expression of the triple fusion reporter did not affect ESC viability, proliferation, and cell differentiation capability, the microarray data provide a cautionary context for interpretation of results. In vitro imaging of induced pluripotent stem (iPS) cells can also provide interesting insights into cellular function. Shambach and colleagues engineered color-coded LVs in which codon-optimized reprogramming factors are coexpressed by a strong retroviral promoter that is rapidly silenced in iPS cells, and then imaged the conversion of the transduced fibroblasts to iPS cells (Warlich et al., 2011). They found that vector silencing occurred concurrent with Oct4–EGFP induction. Approximately 7 days after transduction, the Oct4–EFGP-expressing cells rapidly expanded. Tracking of single cell-derived iPS cell colonies supported the hypothesis that stochastic epigenetic changes are required for reprogramming.

Lentiviral vectors have been used to engineer cell lines for the production of useful proteins of interest. Spencer and colleagues showed that they could improve the production of bioengineered recombinant coagulation factor VIII when produced from an LV-engineered cell line (Spencer et al., 2010). Cell lines secreting high levels of clinically relevant proteins and antibodies could be similarly generated without the laborious effort and time associated with conventional plasmid transfection and selection methods. Particularly relevant is that these proteins exhibit a human glycosylation pattern if produced from lentiviral vector-engineered human cells, which could be functionally or immunologically important in certain circumstances (Jacobs and Callewaert, 2009).

iPS cells have been the focus of attention as a possible source of autologous stem cells for use in regenerative medicine. Pluripotency can be induced in differentiated murine and human cells (e.g., fibroblasts) by transduction of four transcription factors: Oct4, Sox2, Klf4, and c-Myc, as was originally reported using oncoretroviral vectors (Takahashi and Yamanaka, 2006), and later by Jaenisch and colleagues using lentiviral vectors (Brambrink et al., 2008; Welstead et al., 2008). Jaenisch and colleagues extended their initial studies with the development of four transcription factor-expressing vectors expressing from tetracycline/doxycycline-inducible promoters (Brambrink et al., 2008). By switching off inducible gene expression, the iPS-generating genes could be downregulated to allow the stem cells to revert to a normal differentiated state. Repeated switching of iPS transcriptional factor gene expression enabled subsequent generation of secondary iPS cells at a frequency much higher than the initial conversion rate (Hockemeyer et al., 2008; Maherali et al., 2008). One of the limitations of using multiple vectors for cell reprogramming is the high number of vector integrants, which limits their therapeutic development. Hence, several research groups have shown the generation of iPS cells with a single lentiviral vector that comprises all four transcription factors expressed from a single transcript, by using 2A peptide sequences (Welstead et al., 2008; Sommer et al., 2009). However, retention of the iPS-generating factors in the cell population remained undesirable and Cre–loxP sites were later engineered into the 3′ LTR of the vector so that the iPS four-gene cassette could be removed from the cells in the presence of Cre after iPS cell generation (Chang et al., 2009). Although the current trend is to generate iPS cells without resorting to viral vectors, nevertheless LV-mediated generation of iPS cells remains desirable because of the high transduction efficiency afforded by LVs. This is important for the generation of iPS cell clones at a satisfactory frequency (Patel and Yang, 2010; Montserrat et al., 2011). Indeed, Morrisey and colleagues recently demonstrated rapid and efficient reprogramming of mouse and human somatic cells into an iPS cell state with LVs expressing the miRNA 302/367 cluster (Anoke-Danso et al., 2011). Although iPS cells show tremendous potential for regenerative medicine, they can also be used to study stem cell biology and understand disease processes, and as a cellular platform for pharmacological and toxicity testing (Lian et al., 2010). Beyond in vitro applications, LVs can also be used to improve the safety of cells used for therapeutic purposes by introducing safety genes that kill transduced cells after induction, usually by exposure to a prodrug in the case of an adverse event, or for a desired effect (Neschadim et al., 2007).

Animal Model Applications

Transgenic animals are essential research tools, whether they are used to understand gene function or biological processes, as preclinical models for human diseases, or for the validation of drug targets. They have been generated by the direct injection of plasmid DNA into the pronucleus of fertilized oocytes, a method that has largely remained unchanged for more than three decades (Gordon et al., 1980). Lentiviral vectors have emerged as an attractive alternative, because of their high gene transfer efficiencies into zygotes or early progenitor cells from a wide range of species including mice, rats, pigs, cows, monkeys, and birds (Singer and Verma, 2008; Pfeifer and Hofmann, 2009). Transgenic animals generated with lentiviral vectors have also shown higher transgene expression levels and increased survival. They can be bred for multiple generations with no observable adverse effects (Kvell et al., 2010; Reichenbach et al., 2010). Reichardt and colleagues developed transgenic rats that expressed an insulin receptor-specific shRNA transcribed from the Tet-inducible promoter (Herold et al., 2008). Induction of shRNA expression resulted in ablation of insulin receptor protein expression and dysregulation of blood glucose levels. The phenotype was shown to be reversible, with discontinuation of induction leading to insulin reexpression and remission of diabetic symptoms. Regulated transgenic models will prove to be most useful for experimentation purposes because the transgene would not be expressed during development, avoiding potentially toxic or unaccounted-for effects. More recently, LV transgenic animals have been generated from existing knockout animal models to improve their experimental utility. Bernad and colleagues generated nonobese diabetic–severe combined immunodeficient (NOD/SCID) transgenic mice that expressed human granulocyte-macrophage colony-stimulating factor (hGM-CSF), resulting in mice that improve engraftment of transplanted human hematopoietic cells (Punzon et al., 2004). Where conventional methods would have proven difficult, LV transgenic NOD/SCID embryos were generated with 68% efficiency and with 100% penetrance. The use of lentiviral vectors to “humanize” existing mouse models with cytokines and other factors could prove a powerful tool for improvement of human–mouse chimeric animal models. Other important applications of lentiviral vector-mediated transgenesis include the development of animals of agricultural interest that resist disease and increase their productivity (Hofmann et al., 2003; Niemann et al., 2005; Niemann and Kues, 2007).

One of the limitations of transgenic animal models is that the transgene is present in every cell of the animal. Expression of the transgene either occurs in every cell, or in every cell of a particular cell type, if tissue-specific promoters and expression-limiting microRNAs are used. However, disease processes are generally first limited to a few cells from a specific cell type. Lentiviral vectors can be used to mimic such processes by being used directly in animals. For example, Verma and colleagues reported the induction of glioblastoma tumors in adult immunocompetent mice by injecting Cre–loxP-controlled lentiviral vector expressing the Ras and AKT oncogenes (Marumoto et al., 2009). Fewer than 60 glial fibrillary acid-positive protein cells were needed to be transduced to generate the tumors in mice. Transplantation of the glial tumor cells into naive immunocompetent recipient mouse brain resulted in the formation of glioblastoma multiforme-like tumors. This result has tremendous implications for cancer research because fully immunocompetent animal models for cancer can be made simply by the direct injection of LV-expressing oncogenes into appropriate tissues of mice and other animals. Such relevant animal models can also provide greater insights into the pathogenesis of disease processes. Extending the above-described study, Verma and colleagues found that the glioblastoma mice contained tumor-derived endothelial cells and that hypoxia was important in the differentiation of tumor cells into endothelial cells. These tumor-derived endothelial cells were found to be resistant to an anti-vascular endothelial growth factor (VEGF) inhibitor, suggesting that these cells are important in the resistance to anti-VEGF therapy and consequently should be considered a potential target for glioblastoma therapy (Soda et al., 2011).

An important pharmacologic application of animal models is their use for validation of drug targets. Knocking out drug–target gene function in transgenic mice, and showing lack of drug effect when administered to these animals, validates the specificity of the drug and identifies any off-target effects. However target validation can also be performed to confirm the beneficial effects of targeting a specific pathway involved in a disease process. For example, tumor necrosis factor (TNF)-α is upregulated in psoriatic skin and potentially represents a target for psoriasis treatment. However, because TNF-α mRNA is not increased in psoriatic skin cells, it remains unclear whether intervention strategies based on targeting TNF-α with siRNA are therapeutically relevant. To determine the relevance of TNF-α as a target, Mikkelsen and colleagues (Jakobsen et al., 2009) screened a panel of LV shRNAs targeting human TNF-α mRNA and identified the most potent inhibitor. This LV-TNF-α shRNA was assessed in a psoriasis xenograft transplantation model and shown to ameliorate the psoriasis phenotype by reducing epidermal thickness, normalizing skin morphology, and reducing levels of TNF-α mRNA levels in skin biopsies 3 weeks after a single injection of the vector. The findings validated TNF-α mRNA as a target molecule for siRNA-based treatment of psoriasis.

Summary and Future Prospects

Since their first description, pseudotyped lentiviral vectors have become highly used gene transfer vehicles in multiple areas of basic and applied research. They have been validated as highly efficient and robust mediators for gene transfer in a wide variety of in vitro and in vivo settings. Studies in animal models examining the safety of lentiviral vectors and human clinical trials have largely been reassuring, although vector configuration is important and additional studies are required to confirm these early results. Further improvements in vector design and manufacture should help ease lingering concerns. The continued advances in genomics may result in the identification of strategies to further improve their safety, perhaps by targeting to specific regions of the chromosome. As the functions of genes and their targets are identified at an accelerated pace, LVs will play an increasingly important role in their gene transfer and application. Advances in cell engineering, particularly iPS cells, will be enhanced when combined with genes or shRNA that improve their function or safety. For example, the safety of potentially oncogenic, pluripotent stem cells could be dramatically improved if genetically modified to contain safety genes. Beyond laboratory applications, the efficient generation of transgenic animals will be important to meet the growing global demand for food. Improvements in the productivity of cell substrates for biologics manufacturing will become more important as pressure mounts to decrease the cost of health care. The successes of gene therapy in the clinic are obviously exciting. As we incorporate an increasing number of genes, elements, methods, devices, and cell targets into our lentiviral vector gene transfer paradigm, we see the promise of fresh opportunities for application of this remarkable technology.

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