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Virus-based vectors for gene expression in mammalian cells: Adenovirus
S.C. Makrides (Ed.) Gene Transfer and Expression in Mammalian Cells ß 2003 Elsevier Science B.V. All rights reserved
CHAPTER 3.5
Virus-based vectors for gene expression in mammalian cells: Adenovirus Denis Bourbeau1, Yue´ Zeng1, and Bernard Massie1,2,3 1
Institut de Recherche en Biotechnologie, Conseil National de Recherche Canada, 6100 Avenue Royalmount, Montre´al, QC, Canada H4P 2R2; Tel.: þ 1 (514) 496-6131; Fax: þ 1 (514) 496-5143 E-mail: [email protected] 2 INRS-IAF, Universite´ du Que´bec, Laval, QC, Canada H7N 4Z3 3 De´partement de Microbiologie et Immunologie, Faculte´ de Me´decine, Universite´ de Montre´al, Montre´al, QC, Canada H3C 3J7
1. Introduction Adenoviruses (Ad) were first described in the 1950s as causal agents of upper respiratory tract infections and were subsequently associated with only minor pathologies. There are more than 100 Ad serotypes currently identified both in mammals and birds. The Ad virion is a 70–100 nm icosahedral particle composed of 252 capsomers and containing a double-stranded DNA linear genome of 25–45 kb depending on the serotype. Their biology and molecular structure were more extensively characterized using human Ads from serotypes 2 and 5 [1]. As a result, the majority of adenoviral vectors (AdV) were derived from these serotypes. Following receptor interactions, the virus is internalized by means of clathrindependent endocytosis and is rapidly transported to the nucleus. The Ad2 genome is shown schematically in Fig. 1A. It contains cis-acting elements at each end, the inverted terminal repeats (ITRs), which are involved in DNA replication and packaging (at the left ITR). Viral genes are expressed in two phases delineated by the onset of DNA replication; the early phase, which lasts about 6–8 h, and the late phase, which culminates around 30 h post-infection with the production of more than 104 infectious virions per cell. Genes from the early phase are subdivided into six transcription units: E1A, E1B, E2A, E2B, E3, and E4. Each region gives rise to a set of alternatively spliced transcripts from which several proteins are synthesized, whereas two delayed early units produce only one protein, respectively, pIX and IV2a. The roles of the early gene products can be generalized as follows. The E1 genes are involved essentially in activating expression and diverting the cellular machinery toward viral DNA replication and preventing apoptosis. The E2 region bears genes directly involved in viral DNA replication. The genes from E3 region are involved in hampering the host defense system. Finally, the six ORFs forming the E4 region are involved in viral mRNA processing and transport as well as regulation of transcription and apoptosis. Although the function of some of these gene products is unknown, most of them are multifunctional while many share complex interactions with each other. Late genes are expressed from five families of transcripts (L1–5) and encode proteins involved in encapsidation and maturation of viral particles (reviewed in [2]).
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Fig. 1. (A) Simplified schema of the transcriptional map of Ad2/5 (36 kb). Arrows represent viral transcripts, shaded arrows represent transcriptional units. E, early expression; L, late expression. The ITRs and the packaging signal ( ) are indicated. Light-shaded portions of the genome represent DNA that was deleted in several recombinant AdV. (B) Schema of the adenovirus vectors currently reported in the literature. Deletions characterizing the first, second, third generations, and replicative AdV are presented as follows, in order of appearance. 1st generation: E1E3. 2nd generation: (E1, E2B, pTP, E3), (E1, E2A, E3), (E1, E3, E4), (E1, E2A, E3, E4). 3rd generation: all genes deleted, all genes deleted but pTP.
111 In the last two decades Ads have been extensively used for gene transfer into mammalian cells (reviewed in [3]). Several features make AdV attractive vehicles for gene transfer and protein production. First, AdV can easily be produced at high titers; second, they can transduce a large variety of cell types; third, they do not require cell division for transgene expression; and fourth, their genome remains episomal, therefore they do not permanently alter the target cell. AdVs have been extensively engineered in order to be adapted to several applications of gene transfer in mammalian cells and gene therapy in vivo. In this chapter, we aim to provide the reader with an overview of the AdV platforms currently developed, with means to engineer, produce, and characterize them.
2. Adenoviral vectors To effect the transfer of exogenous genes into mammalian cells, the Ad has been modified in order to: (1) eliminate its replication capacity; (2) increase its cloning size capacity; (3) reduce the host immune response; and (4) retarget it to specific cell receptors. The most widely used AdV is the so-called first generation that is deleted in the E1 and the E3 regions. Later, other modifications were made in order to meet specific objectives. The various AdV engineered to date are depicted in Fig. 1B. 2.1. First generation AdV A deletion that removed the E1A and E1B genes distinguishes the first generation AdV from the wild type Ad. The E1 deletion renders the AdV replication dependent on cell lines such as the 293, A549-E1, or PER.C6, which complement the E1 functions [4]. The E3 region is not required for viral production in vitro and thus its deletion increases the cloning capacity without having to complement its function in producing cells. Those vectors can accommodate exogenous DNA of up to 8 kb in size. Construction of first generation AdV is routinely performed by recombination between a digested AdV genome and a transfer plasmid that contains the left arm portion of the AdV, the transgene cassette, and a portion of homologous sequence for recombination to occur. Recombination is performed in mammalian cells, generally in 293 cells, which are readily transfected by the calcium phosphate procedure. The recombination requires the co-transfection of both the transfer plasmid and the viral genome. This procedure, although favored for its simplicity, is inefficient. Recombination is a rare event and incompletely digested AdV may give rise to non-recombinant AdV. Our laboratory is relying on co-expression Replicative AdV: PS, 100K, E1B-55K, E1A promoter substitution. Abbreviations: ITR, inverted terminal repeat; pol, Ad polymerase; pTP, preterminal protein; PS, Ad protease; DBP, DNA-binding protein; E1, E2, E3, E4, early phase transcriptional units; L1, L2, L3, L4, L5, late phase transcriptional units.
112 of the green fluorescent protein (GFP) to select recombinant plaques under the microscope [5]. Several of our transfer vectors carry a cassette, which joins the GFP with the transgenes through an internal ribosome entry site (IRES). In this way, GFP expression is linked to transgene expression, and recombinant plaques can be distinguished from non-recombinant ones. Other strategies have been developed in order to minimize contamination with non-recombinant AdV and to reduce the time required for AdV production. These strategies rely on a viral genome that is carried as a plasmid or cosmid, and recombinant AdV can be produced by several means: (i) subcloning directly the transgene into the plasmid containing the viral genome; (ii) using a recombinase such as Cre or Flp that facilitates specific and efficient introduction of transgenes into the viral genome; or (iii) using bacterial recombination for introduction of the transgene into the viral genome. Strategies of direct cloning into AdV genome serve the purpose of eliminating non-recombinant plaques, therefore plaque purification. Cloning in large plasmids is difficult; thus cosmids were designed to facilitate the subcloning steps [6]. The strategy making use of recombinases replaces the subcloning step through efficient homologous recombination either in bacteria or in mammalian cells. Such strategies require AdV that are engineered to contain recombination sequences, such as loxP or frt, which are substrates for Cre and FLp, respectively, and cell lines or bacteria expressing the specific recombinase [6,7]. Recently, a novel strategy based on positive selection was developed for producing recombinant AdV. The strategy consists of inserting the Ad protease gene in the shuttle plasmid, to complement a protease (PS) deletion in the virus backbone used for recombination. Since the virus backbone cannot form viral particles in the absence of the PS, the only infectious particles produced are those that have recombined with the complementing shuttle plasmid. Thus, following transfection of permissive cells with the shuttle plasmid and infection with an AdV deleted for its PS gene, pure recombinant plaques are produced within 2 weeks with an efficiency of 100%. For the construction of recombinants one at a time, this positive selection method is at least as efficient as the best methods developed so far. Furthermore, this represents the first method allowing for easy construction of AdV libraries with high diversities [8]. 2.2. Second generation AdV First generation AdV can sustain transgene expression for years in post-mitotic cells of immunodeficient animals. However, their expression will persist only a few weeks in actively growing cells, due to the dilution of the viral genome by cell division, or in immunocompetent animals, due to the host immune system that destroys the transduced cells. Despite the absence of E1 genes required to initiate expression of viral genes, de novo viral gene expression occurs to some extent. Viral proteins are then exposed to the cell surface and elicit an immune response. In order to minimize this unwanted expression, AdV have been further crippled by deletion of genes in the E2 or E4 regions. The production of these second generation AdV has required the
113 development of complementation cell lines expressing the corresponding deleted genes. Deletions were engineered in E2A, E2B, DNA polymerase and/or preterminal protein (pTP), as well as E4, and in most cases in vivo studies showed significant reduction of the immune response. In the case of E2B, the deletion of either the polymerase or pTP alone decreased hepatic toxicity and prolonged vector stability, while combining both deletions resulted in further improvement [9]. In the case of E4 deletions, the generation of Ad E1 E2A E4 demonstrated that further crippling of AdV by removing E4 genes reduced vector toxicity but also reduced vector stability [10]. In fact, it appears that ORF3 is required for efficient expression of transgene under the control of several promoters used in AdV, particularly the CMV promoter [11,12]. The mechanism responsible for the reduced expression and stability of second generation AdV deleted in E4 remains to be elucidated. Nonetheless, most studies using E4-deleted AdV have reported decreased transgene stability, unless the ORF3 or ORF6 have been reintroduced. Moreover, reintroducing ORF3 in E4-deleted AdV restores its stability while retaining a reduced toxicity. On the other hand, ORF6 reintroduction restores both stability and hepatotoxicity [13]. It is noteworthy that either ORF3 or ORF6 can complement the complete E4 deletion. 2.3. Third generation AdV Because second generation AdV were still sub-optimal in escaping the immune response, and there was a need for larger exogenous DNA insert capacity, the more drastic approach of eliminating all viral genes was undertaken. Fully deleted vectors have been given several names: third generation, helper-dependent, high capacity, or gutless. These AdV offer three main advantages: (1) no expression of viral genes is possible, thus reducing the immune response; (2) a cloning capacity of up to 36 kb; and (3) they retain all of the advantages of first generation AdV with respect to transduction efficiency. However, they are more difficult to produce since it is almost impossible to generate a stable cell line that would complement all of the Ad genes, due to the toxicity associated with the over-expression of most of the Ad proteins. The helper functions required for the complementation of third generation AdV are most often provided by co-infection with a helper virus, but they can also be provided by co-transfection of the adenoviral genome as plasmid [14], or through a baculovirus/Ad hybrid [14–16]. The dependency on a helper Ad results in decreased viral yields and contamination with helper viruses. A few strategies have been designed to overcome these drawbacks. The strategy that is currently favored is to remove the helper virus-packaging signal through Cre/loxP recombinase [17], and more recently the Flp/frt recombinase system [18]. These systems require that third generation AdV be amplified with a helper Ad bearing loxP or frt sequences flanking its packaging signal. In 293 cells expressing Cre or Flp recombinases, the packaging signal of the helper virus is deleted resulting in viral DNA that still can produce viral proteins, but cannot be packaged into viral particles. Although the cleavage by Cre or FLP is not 100% and some helper viruses are still packaged, this procedure
114 reduces contamination to less than 0.01% after CsCl purification in the best stocks. While helper AdV are a very small proportion of the preparation, high-dose injections such as 1012 particles would still deliver more than 108 contaminating particles. This might be sufficient to elicit an immune response. Therefore, further improvement in third generation AdV purity is required. Another issue about the production of third generation AdV is the requirement of an optimal genome size of 26–38 kb for efficient particle formation. In order to generate optimized third generation AdV, the large amount of deleted DNA must be replaced with stuffer DNA that is carefully chosen. Thus, the stuffer DNA should have the following desirable characteristics: (1) it should be preferentially of mammalian origin; (2) it should be free of repeats, retrovirus elements, and genes; (3) contiguous human DNA should be fragmented to prevent integration; and (4) a matrix attachment region might confer stability [19]. Other cis-acting elements, such as a 400-bp fragment from the right end of Ad, which appears to confer growth advantage, can contribute to improved yields of gutless AdV [19]. While efforts are still ongoing to further optimize their production, several studies performed with third generation AdV have provided very promising results in reducing immune response and prolonging stability. The hurdles remain essentially in the production of clinical grade third generation viruses due to the complexity of the process. 2.4. Replicative AdV While a major concern in the use of AdV for gene replacement therapy is reduction of toxicity and host immune response, there are applications, such as vaccination or cancer gene therapy, where toxicity and immunogenicity are rather an asset. For such applications the increased transgene expression through an increase in copy number following viral DNA replication can also be advantageous. This can be done by deleting genes that do not affect DNA replication, such as the PS or the 100K (late genes), while interfering with the production of infectious particles [20,21]. Although the full potential of such AdV has yet to be demonstrated, they could have applications for recombinant protein production, vaccination, cancer gene therapy, and production of adeno-associated viruses (AAV). Other types of replicative AdV were developed more specifically for cancer gene therapy. Because transduction efficiency of 100% is not achievable, yet complete eradication of tumor cells is required for successful treatment; the ability of transduced tumor cells to kill bystander tumor cells is of paramount importance. One approach to achieving this goal is to engineer AdV that can replicate and form mature particles only in tumor cells. This was achieved using AdV deleted either in the E1B-55 kDa and/or the E1B-19 kDa gene. Thus, after infecting a few tumor cells, the progeny of conditional replicative oncolytic AdV can spread and destroy neighboring tumor cells while sparing normal cells. Other strategies rely on a tumorspecific promoter to drive E1A gene expression [22]. The above strategies have also been combined with cytokine or suicide genes to further improve the efficacy of conditional replicative AdV.
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3. Gene delivery 3.1. Cell entry pathway Effective vectors are those which deliver the transgene specifically to the appropriate tissues. Strategies for targeting AdV to specific cell types became possible following the improved understanding of the viral entry mechanisms, including the identification of the viral proteins and cellular receptors that are involved in virus–cell interactions. 3.1.1 Ad capsid proteins involved in virus entry Two capsid proteins, the fiber and the penton base, play essential roles in virus–cell interaction and virus internalization. Five penton base subunits are associated with each fiber, itself composed of a homotrimer of fiber proteins. The fiber is responsible for the assembly and stabilization of the virion. It can be divided into three domains with distinct functions: (1) the short amino-terminal tail is involved in its association with the penton base protein through an FNPVYP motif. This interaction anchors the fiber to the Ad capsid. Also located in this domain is a nuclear localization signal (KRXR), which directs the intracellular trafficking of newly synthesized fibers to the cell nucleus where Ad particles are assembled. (2) The central shaft domain, whose length varies among different serotypes, is characterized by sequence repeats of approximately 15 residues. The shaft domain, which makes the carboxy terminal end of the fiber extending away from the virion, therefore provides optimal condition for fiber–receptor recognition. (3) The carboxy terminal knob domain forms an sandwich structure, which contains two anti-parallel -sheets named R and V. These sheets are each composed of four strands linked by loops in which several residues are involved in the binding of the virus to the coxsackie virus and Ad receptor (CAR). Another distinct function carried out by the knob domain is the initiation of fiber trimerization. The penton base subunit contains a flexible loop structure consisting of two stretches of helices. The loop of the five penton base units forms a protrusion on the protein surface, exposing an arginine–glycine–aspartic acid (RGD) motif (aa 340–342), which interacts with the cellular integrin v, and a leucine–aspartic acid–valine (LDV) sequence (aa 287–289) also identified as an integrin-binding motif. In addition, the penton base plays an important role in the internalization of viral particles and release of capsids from the endosome. 3.1.2 Cell receptors CAR, a 46-kDa transmembrane glycoprotein widely expressed in different tissues, is the primary receptor for Ad transduction. It belongs to the immunoglobulin (Ig) superfamily and contains two extracellular immunoglobulin-like domains, a hydrophobic transmembrane region and an intracellular cytoplasmic domain. Functional analyses of recombinant forms of CAR have demonstrated that the first immunoglobulin-like domain is sufficient for Ad binding. It should be noted that certain subgroup B or D Ads do not use CAR as receptor for their entry. Also, an
116 sialic acid residue of some glycoproteins is used by Ad37 as receptor for their cell entry [23]. Interaction of the fiber with CAR leads to virus attachment onto the cell surface, but, in general, this is not sufficient for rapid virus uptake. It is the association of the cellular integrins v3 or v5 with the viral penton base that facilitates virus internalization. Moreover, fiber-less particles have been demonstrated to keep their infectious capacity for monocytic cells via an integrin-dependent pathway [24]. Recent studies have suggested that heparan sulfate glycosaminoglycans are sufficient to mediate Ad2/5 binding on the cell surface and infection [25]. The 2 domain of the major histocompatibility complex class I protein (MHC-I) is also reported to function as an alternative receptor [26]. However, when CAR and MHC-I proteins were co-expressed on the same cell, only CAR had high affinity for the Ad5 fiber. MHC-I-dependent Ad5 attachment may only occur when CAR expression is low. 3.1.3 Mechanisms of early steps of cell infection by Ad Cellular uptake of the Ad particle is a two-step process: (1) an initial interaction of the fiber with cellular receptors like CAR or MHC-I [27]; and (2) the internalization of the virus mediated by the interaction of the RGD motif of the penton base with other cellular receptors, such as the integrins v3 and v5. This latter interaction triggers the phosphorylation of several signaling molecules, and induces a signaling cascade that promotes actin polymerization, which provides the mechanical force necessary for the internalization of clathrin-coated pit containing AdV. Viral proteins begin to be dismantled during the endocytotic uptake from the plasma membrane and during subsequent changes in the endosomes. After the endosomal membrane is lysed, the uncoated particle escapes to the cytoplasm with the help of the penton base protein, and the core is translocated to the nucleus through nuclear pores. The Ad genome then enters the nucleoplasm where viral replication takes place. 3.2. Targeting of AdV Active research is ongoing to develop targeting vectors to selectively transduce the tissue of interest for the following reasons: the Ad wide tropism leads to indiscriminate transduction of both target and bystander cells. Transduction of nontarget cells not only dilutes the amount of therapeutic viruses delivered to target cells but also results in undesirable transgene expression. Moreover, because some cells have no or low levels of CAR, targeting AdV to a specific cellular receptor would overcome the problem of poor transduction efficiency. Finally, preventing AdV interaction with antigen-presenting cells should reduce the humoral and cytotoxic T lymphocyte response caused by AdV in vivo. Since the fibers of different Ad serotypes display various amino acid sequences, it was suggested that they might recognize different receptors and consequently have different tropism. Therefore, several studies were undertaken using either chimeric
117 fibers or exchanging fibers from different serotypes in order to alter AdV tropism. For example, Shayakhmetov et al. [28] demonstrated that the chimeric vector Ad5/ F35 can successfully infect human CD34 þ cells by CAR- and v integrinindependent pathways. However, effective targeting requires the abolition of vector interaction with its natural receptors, and the redirection of the vector to another type of receptor, which is specific to the target cells. Two strategies have been developed to realize this goal: (1) a conjugate system that allows AdV interaction with bridging molecules for the binding to a new receptor; and (2) a genetic approach in which the virus capsid proteins are modified to recognize only the target receptor (Fig. 2A).
Fig. 2. (A) Schema depicting two strategies to retarget AdV. Left, the natural tropism of AdV and its inability to interact with certain target cells. Middle, the use of a bifunctional molecule bridging the virus to the target cell. Right, the genetic modification of AdV as the alternate strategy. (B) Special packaging cells need to be engineered for production of genetically modified AdV that may fail to infect ‘‘normal’’ packaging cells.
118 3.2.1 Conjugate targeting system This system makes use of a bispecific molecule, which binds to the AdV and to the targeted receptor. The AdV specificity is usually provided by a soluble form of CAR, the binding domain of MHC, or neutralizing antibodies against one of the capsid protein. Other conjugation strategies include biotinylation, and coating with polyethyleneglycol (PEGylation) or a multivalent hydrophilic polymer. The cellular specificity can be generated with an antibody or a high-affinity ligand specific for a receptor or an adhesion molecule expressed on the targeted cell surface, such as CD40, v integrin, the receptors for fibroblast growth factor (FGF), tumor necrosis factor (TNF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and several tumor markers. The bispecific molecules were made by chemically linking two molecules, or by engineering recombinant fusion proteins. Any receptor can be targeted with the above strategies because any high-affinity antibody can be used as ligand. An additional advantage of the PEGylation and multivalent polymeric modification of AdV is that vectors are protected against neutralizing antibodies, thus avoiding some of the immune responses. However, some limitations remain to be overcome: there are several steps involved in the largescale production and purification of AdV and bispecific molecules in the development of stable forms of the complexes for storage and for intravenous administration. Thus, the development of other approaches, such as genetic retargeting, may be more promising. 3.2.2 Genetic targeting system This approach relies on genetic modifications to incorporate targeting ligands to the coat proteins of AdV. One challenge is to determine which locations are appropriate for incorporating high-affinity ligands. Wickham et al. [29] have constructed AdV whose fibers contain an RGD motif or a polylysine in its C-terminus. These viruses increased transduction in multiple cell types lacking high levels of CAR. The HI loop of fiber, the RGD loop of penton base, and an exposed loop of hexon were also chosen for the insertion of high-affinity peptides. Modifications in these locations were demonstrated to lead to virus attachment and cell entry in a CAR-independent manner. Another issue concerns the size of the peptide to be inserted. The incorporation of a ligand into Ad capsid proteins must not alter the conformation of these proteins, and should not adversely affect their other normal functions. Ideal ligands are small peptides specific for the targeted cellular receptor. Published findings suggest that less than 25–30 residues can be inserted into the C-terminus of the fiber, because longer sequences destabilize the fiber trimer [30]. It is also reported that ligands added into the HI loop of the fiber should not exceed 63 amino acids [27]. Full re-targeting requires the elimination of AdV interaction with its natural receptors. Several amino acid residues localized in the knob region of Ad5 fiber, such as S408, P409, K417, K420, K442, Y477, L485, Y491, A494 and A503 are directly involved in fiber–CAR interaction. Mutations of these amino acids and of residues 489–492 lead to a drastic reduction in virus transduction [31]. Several studies have successfully combined CAR-binding ablation and targeting ligand insertion for retargeting. For example, Nicklin et al. [32] have retargeted AdV to endothelial
119 cells by mutating two amino acid residues (S408E and P409A) in the knob region that blocks CAR interaction, and by incorporating the SIGYPLI peptide, which was demonstrated to bind specifically to endothelial cells. Finally, disruption of v integrin at the same time is also critical for fully ablating the natural AdV tropism. The production of a fully ablated AdV in its endogenous receptor interaction requires: (1) the insertion in the Ad capsid protein of a novel ligand unable to bind to any cells; and (2) the use of novel packaging cells expressing a chimeric receptor that allows entry and replication of the modified AdV [33,34]. Therefore, in addition to the targeting ligand incorporated into the AdV capsid for cell-specific transduction, another pseudoreceptor-binding ligand should also be inserted into the vector for entry into the packaging cells (Fig. 2B). This pair of pseudoreceptor–ligand for virus propagation should preferably be completely artificial, otherwise it might compromise the specificity of the retargeting, since any natural analogue could cause binding of the vector to non-target cells in vivo.
4. Gene expression 4.1. High-level expression Besides transduction efficiency, transgene expression efficiency has also been improved. High expression level of recombinant proteins with low AdV multiplicies of infection (MOI) are desirable to minimize negative effects of high MOI on target cells. Strong promoter-enhancer elements usually derived from viruses have been used to construct expression cassettes. For example, the immediate-early human cytomegalovirus (hCMV) promoter has been utilized extensively because of its high transcriptional activity in a wide range of cells. However, AdV containing standard CMV-based expression cassettes produce proteins at suboptimal levels representing no more than 2–3% of total cell protein (TCP). Optimizing the expression cassette by including the Ad tripartite leader and a small intron as in pAdCMV5 improved performance by 6–12-fold, both at low and high MOI, and achieved up to 35% of TCP in AdV-infected cells at high copy number [5,35]. The expression of some proteins may have deleterious effects on the host cell or on Ad production. Inducible gene expression systems (Chapter 22) have been therefore developed to overcome this problem. Regulation of transgene expression in vivo can also improve its safety and efficacy. An ideal inducible system would have low basal expression of transgene without the inducer and very high expression in its presence (reviewed in [36]). The most widely used inducible promoter is the tetracyclinecontrollable transactivator system. A trans-acting factor (tTA) is formed by the fusion of the activation domain of the herpes simplex virus (HSV) protein VP16 with a tetracycline repressor protein from Escherichia coli. The tTA transactivator stimulates transcription from a promoter containing the tetracycline operator sequences (tetO), but the interaction of tTA with tetO is prevented in the presence of tetracycline at concentrations not toxic to eukaryotic cells. The use of an optimized tet-regulated promoter, as in pAdTR5, allows the expression of proteins that are
120 cytotoxic or interfere with Ad replication, at 10–15% TCP levels [5,35]. Recently, a new inducible gene switch was developed in our laboratory using the operator sequences and repressor of a bacterial p-cymeme operon whose DNA-binding can be regulated by cumate. This cumate-inducible system is comparable to the tet system in many respects. Interestingly, the cumate-inducible promoter achieved higher transgene expression than the tet-regulated promoter at lower MOI, thereby minimizing the interference of AdV at higher MOI. Moreover, combining the tet and cumate regulatory elements reduced leakiness while fully maintaining the expression level of the on state. Several other inducible systems have also been tested in AdV. These include the cre-loxP system from bacteriophage P1, the bacteriophage T7 binary system [37], the insect ecdysone system and the human rapamycine-inducible system. These inducible systems have intrinsic advantages and limitations that should be carefully evaluated in comparative studies in order to select the optimal system for a specific application (reviewed in [38]). 4.2. Specific transgene expression Many studies have explored the use of tissue-specific promoters mainly to restrict transgene expression to the target tissues in order to avoid unwanted expression and reduce the immune response against the transgene product. Tissue-specific promoters are also used to transduce particular types of tumor cells in cancer therapy, as discussed above. 4.2.1 Tissue-specific promoters Tissue-specific targeted gene transcription was mainly attempted for expression in the lung, epithelia, liver, pancreas, muscles, neural cells, mammary gland and cardiac cells. For example, the amylase promoter is used to target pancreas cells, truncated muscle creatine kinase (MCK) and the smooth muscle alpha-actin (SMA) promoters for targeting skeletal and smooth muscle cells, ventricle-specific myosin light chain 2, ventricle specific -myosin heavy chain and troponin T promoters are used for cardiac cells, L7/PCP2 promotor for cerebellar Purkinje cells, and synapsin 1, tubulin alpha 1 and neuron-specific enolase (NSE) promoters are used to target neurons. Using silencer elements is another approach for specific transgene expression. For example, elements specific to neurons can selectively repress the transcription of genes in non-neuronal cells so that when they are cloned upstream of the ubiquitous phosphoglycerate kinase promoter, transgene expression is restricted to neurons [39]. 4.2.2 Tumor-specific promoters Transcriptional targeting of tumors has been investigated with promoters known to be particularly active in tumor cells. The use of tumor-specific promoters should result in expression of a therapeutic gene primarily in tumor cells, while marginal expression should be observed in normal cells. There are several promoters that meet this objective: the MUC1/Df3 promoter has been used for breast carcinoma, the alpha fetoprotein (AFP) promoter for hepatomas, the cyclooxygenase-2 (cox-2)
121 promoter for gastrointestinal cancers, the CTP1 promoter for colon cancer, the prostate-specific antigen (PSA), human glandular kallikrein (hk2) and probasin promoters for prostate carcinoma, and the Midkine (MK) promoter for neuroblastoma. Promoters that are particularly efficient in actively dividing cells are also useful for cancer targeting, since in adults, few cells other than tumors are replicating. The E2F-responsive promoter E2F-1, is one of them. It allows eradication of established gliomas with significantly less toxicity to normal tissue than constitutive promoters [3]. A similar approach made use of the hexokinase type II promoter to preferentially drive the expression of a toxic gene in a variety of cancer cell lines [3]. The human telomerase reverse transcriptase (hTERT) promoter was also shown to be strong and preferentially active in tumor cells. This promoter enabled induction of the Bax gene to elicit tumor-specific apoptosis in vitro, and in vivo it caused the suppression of tumor growth in nude mice while preventing the cytotoxic effects of the Bax gene to normal cells [40].
5. Production and analyses of viral particles First-generation AdV are commonly produced in 293 cells that express the E1 genes from a large fragment of the left end of the Ad genome integrated in their chromosomal DNA. However, homologous sequences between AdV and the genome of 293 cells allow for occasional recombination leading to the generation of replication-competent AdV (RCA). This problem has been solved by the development of new E1-complementing cell lines, which do not share homologies with E1-deleted AdV [3,4]. Pharmaceutical and commercial applications of AdV require virus production on a large-scale. This has been facilitated by the development of processes allowing the production of AdV in suspension and in serum-free cultures [41]. Following production, AdV lysates are prepared by three freeze-thaw cycles and removal of cellular debris. These viral crude lysates can readily be used for in vitro studies. However, for many applications it is preferable to purify and concentrate the AdV particles. This is most often done by CsCl gradient purification, which relies on the specific buoyancy of AdV. However, this procedure is time-consuming and difficult to scale-up, necessitating the development of other purification protocols based on chromatography [42]. For the production of clinical-grade material, several challenges remain to be addressed to make the process economically viable and fully compliant with regulatory requirements. Thus, the yield, homogeneity, lot consistency, and a reliable characterization of the vector particles and infectivity need to be addressed. Regarding consistency and comparability of AdV production among different laboratories, the establishment of a standard sample and of standard operating procedures (SOPs) was awaited. The Adenovirus Reference Material Working Group (ARMWG) has established such standards. A wild-type Ad5 will be available from the ATCC in 2002, and SOPs for evaluation of viral particles and infectious particles are published at http://www.wilbio.com. The establishment of this standard
122 is crucial to the comparability of AdV quantification across different laboratories. Current titration measures have different efficiency and are all extremely sensitive to absorption conditions (time, volume, agitation). Moreover, the intrinsic sensitivity of each assay varies within one order of magnitude (TCID50, plaque assay, transduction units). A report on the AdV standards has been published [43]. Finally, AdV need to be stored under conditions that ensure their stability over time. Croyle et al. [44] have developed a formulation for the long-term storage of lyophilized AdV at room temperature without loss of virus stability. Recently, the ARMWG has proposed a rather simple formulation that performed well for a period of over 6 months. This formulation is composed of 20 mM Tris pH 8.0, 25 mM NaCl, 2.5% glycerol, and is suitable for the stable storage of AdV at a concentration below 5 1011 particles [45].
6. Conclusion The biological features of AdV have contributed to their popularity as tools for functional studies and gene therapy applications as demonstrated by the thousands of reports on AdV in recent years. Because efficacy and specificity is of paramount importance in gene therapy success, considerable efforts have been invested on their improvement. Although several challenges in targeting and reducing the host immune response remain to be addressed, current progress has armed molecular biologists with a wealth of AdV adapted to numerous applications. Moreover, new developments in the construction of Ad-retro, Ad-AAV or Ad-EBV hybrid viruses will further expand their use in therapy that requires long-term transgene expression. Finally, the recent development of Ad-based libraries [8] will lead to new applications yet to be explored.
Acknowledgements We thank Re´nald Gilbert for critical reading of the manuscript. This is a NRC publication #37701.
Abbreviations AAV Ad AdV ARMWG CAR CMV CPE GFP
adeno associated virus adenovirus adenovirus vector(s) adenovirus reference material working group coxsackie virus and adenovirus receptor cytomegalovirus cytopathic effect green fluorescent protein
123 IRES ITR MHC MOI ORF pTP PS RCA SOP TCP tetO tTA
internal ribosome entry site inverted terminal repeats major histocompatibility complex multiplicity of infection open reading frame preterminal protein adenovirus protease replication-competent adenovirus standard operating procedure total cell protein tetracycline operator tetracycline trans-activator
References 1. Shenk, T.E. (2001) In: Knipe, D.M. (ed.) Field’s Virology, Lippincott Williams & Wilkins, Philadelphia, 4th ed, 2265–2300. 2. Russell, W.C. (2000) J. Gen. Virol. 81, 2573–2604. 3. Oualikene, W. and Massie, B. (2000) In: Al-Rubeai, M. (ed.) Cell Engineering, Kluwer Academic Publishers, London, pp. 80–1542. 4. Fallaux, F.J., Bout, A., van der Velde, I., van den Wollenberg, D.J., Hehir, K.M., Keegan, J., Auger, C., Cramer, S.J., van Ormondt, H., van der Eb, A.J., Valerio, D., Hoeben, R.C. (1998) Hum. Gene Ther. 9, 1909–1917. 5. Massie, B., Mosser, D.D., Koutroumanis, M., Vitte´-Monty, I., Lamoureux, L., Couture, F., Paquet, L., Guilbault, C., Dionne, J., Chahla, l., Jolicoeur, P., Langelier, Y. (1998) Cytotechnology 28, 53–64. 6. Danthinne, X. and Imperiale, M.J. (2000) Gene Ther. 7, 1707–1714. 7. Mizuguchi, H., Kay, M.A., and Hayakawa, T. (2001) Adv. Drug Deliv. Rev. 52, 165–176. 8. Elhai, S.M., Oualikene, W., Naghdi, L., O’Connor-McCourt, M., and Massie, B. (2002) Gene Ther. 9, 1238–1246. 9. Hodges, B.L., Serra, D., Hu, H., Begy, C.A., Chamberlain, J.S., and Amalfitano, A. (2000) J. Gene Med. 2, 250–259. 10. Andrews, J.L., Kadan, M.J., Gorziglia, M.I., Kaleko, M., and Connelly, S. (2001) Mol. Ther. 3, 329–336. 11. Armentano, D., Smith, M.P., Sookdeo, C.C., Zabner, J., Perricone, M.A., St George, J.A., Wadsworth, S.C., and Gregory, R.J. (1999) J. Virol. 73, 7031–7034. 12. Grave, L., Dreyer, D., Dieterle, A., Leroy, P., Michou, A.I., Doderer, C., Pavirani, A., Lusky, M., and Mehtali, M. (2000) J. Gene Med. 2, 433–443. 13. Christ, M., Louis, B., Stoeckel, F., Dieterle, A., Grave, L., Dreyer, D., Kintz, J., Ali Hadji, D., Lusky, M., Mehtali, M. (2000) Hum. Gene Ther. 11, 415–427. 14. Kochanek, S. (1999) Hum. Gene Ther. 10, 2451–2459. 15. Morsy, M.A. and Caskey, C.T. (1999) Mol. Med. Today 5, 18–24. 16. Cheshenko, N., Krougliak, N., Eisensmith, R.C., and Krougliak, V.A. (2001) Gene Ther. 8, 846–854. 17. Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. (1996) Proc. Natl. Acad. Sci. USA 93, 13565–13570. 18. Umana, P., Gerdes, C.A., Stone, D., Davis, J.R., Ward, D., Castro, M.G., and Lowenstein, P.R. (2001) Nat. Biotechnol. 19, 582–585. 19. Sandig, V., Youil, R., Bett, A.J., Franlin, L.L., Oshima, M., Maione, D., Wang, F., Metzker, M.L., Savino, R., Caskey, C.T. (2000) Proc. Natl. Acad. Sci. USA 97, 1002–1007. 20. Oualikene, W., Lamoureux, L., Weber, J.M., and Massie, B. (2000) Hum. Gene Ther. 11, 1341–1353.
124 21. Hodges, B.L., Evans, H.K., Everett, R.S., Ding, E.Y., Serra, D., and Amalfitano, A. (2001) J. Virol. 75, 5913–5920. 22. Alemany, R., Balague, C., and Curiel, D.T. (2000) Nat. Biotechnol. 18, 723–727. 23. Arnberg, N., Edlund, K., Kidd, A.H., and Wadell, G. (2000) J. Virol. 74, 42–48. 24. Von Seggern, D.J., Chiu, C.Y., Fleck, S.K., Stewart, P.L., and Nemerow, G.R. (1999) J. Virol. 73, 1601–1608. 25. Dechecchi, M.C., Melotti, P., Bonizzato, A., Santacatterina, M., Chilosi, M., and Cabrini, G. (2001) J. Virol. 75, 8772–8780. 26. Hong, S.S., Karayan, L., Tournier, J., Curiel, D.T., and Boulanger, P.A. (1997) EMBO J. 16, 2294–2306. 27. Krasnykh, V.N., Douglas, J.T., and van Beusechem, V.W. (2000) Mol. Ther. 1, 391–405. 28. Shayakhmetov, D.M., Papayannopoulou, T., Stamatoyannopoulos, G., and Lieber, A. (2000) J. Virol. 74, 2567–2583. 29. Wickham, T.J., Tzeng, E., Shears, L.L., 2nd, Roelvink, P.W., Li, Y., Lee, G.M., Brough, D.E., Lizonova, A., and Kovesdi, I. (1997) J. Virol. 71, 8221–8229. 30. Curiel, D.T. (1999) Ann. N.Y. Acad. Sci. 886, 158–171. 31. Kirby, I., Davison, E., Beavil, A.J., Soh, C.P., Wickham, T.J., Roelvink, P.W., Kovesdi, I., Sutton, B.J., and Santis, G. (2000) J. Virol. 74, 2804–2813. 32. Nicklin, S.A., Von Seggern, D.J., Work, L.M., Pek, D.C., Dominiczak, A.F., Nemerow, G.R., and Baker, A.H. (2001) Mol. Ther. 4, 534–542. 33. Douglas, J.T., Miller, C.R., Kim, M., Dmitriev, I., Mikheeva, G., Krasnykh, V., and Curiel, D.T. (1999) Nat. Biotechnol. 17, 470–475. 34. Einfeld, D.A., Brough, D.E., Roelvink, P.W., Kovesdi, I., and Wickham, T.J. (1999) J. Virol. 73, 9130–9136. 35. Massie, B., Couture, F., Lamoureux, L., Mosser, D.D., Guilbault, C., Jolicoeur, P., Belanger, F., and Langelier, Y. (1998) J. Virol. 72, 2289–2296. 36. Mullick, A. and Massie, B. (2000) In: Speir, R.E. The Encyclopedias of Cell Technology, Wiley Biotechnology Encyclopedias, London, pp. 1140–1164. 37. Tomanin, R., Bett, A.J., Picci, L., Scarpa, M., and Graham, F.L. (1997) Gene 193, 129–140. 38. Fussenegger, M. (2001) Biotechnol. Prog. 17, 1–51. 39. Millecamps, S., Kiefer, H., Navarro, V., Geoffroy, M.C., Robert, J.J., Finiels, F., Mallet, J., and Barkats, M. (1999) Nat. Biotechnol. 17, 865–869. 40. Gu, J., Kagawa, S., Takakura, M., Kyo, S., Inoue, M., Roth, J.A., and Fang, B. (2000) Cancer Res. 60, 5359–5364. 41. Cote, J., Garnier, A., Massie, B., and Kamen, A. (1998) Biotechnol. Bioeng. 59, 567–575. 42. Huyghe, B.G., Liu, X., Sutjipto, S., Sugarman, B.J., Horn, M.T., Shepard, H.M., Scandella, C.J., and Shabram, P. (1995) Hum. Gene Ther. 6, 1403–1416. 43. Hutchins, B. (2002) BioProcessing 1, 25–28. 44. Croyle, M.A., Cheng, X., and Wilson, J.M. (2001) Gene Ther. 8, 1281–1290. 45. Hoganson, D., Ma, J.C., Asato, L., Ong, M., Printz, M.A., Huyghe, B.G., Sosnowski, B.A., and D’Andrea, M.J. (2002) BioProcessing 1, 43–48.
CHAPTER 3.5
Virus-based vectors for gene expression in mammalian cells: Adenovirus Denis Bourbeau1, Yue´ Zeng1, and Bernard Massie1,2,3 1
Institut de Recherche en Biotechnologie, Conseil National de Recherche Canada, 6100 Avenue Royalmount, Montre´al, QC, Canada H4P 2R2; Tel.: þ 1 (514) 496-6131; Fax: þ 1 (514) 496-5143 E-mail: [email protected] 2 INRS-IAF, Universite´ du Que´bec, Laval, QC, Canada H7N 4Z3 3 De´partement de Microbiologie et Immunologie, Faculte´ de Me´decine, Universite´ de Montre´al, Montre´al, QC, Canada H3C 3J7
1. Introduction Adenoviruses (Ad) were first described in the 1950s as causal agents of upper respiratory tract infections and were subsequently associated with only minor pathologies. There are more than 100 Ad serotypes currently identified both in mammals and birds. The Ad virion is a 70–100 nm icosahedral particle composed of 252 capsomers and containing a double-stranded DNA linear genome of 25–45 kb depending on the serotype. Their biology and molecular structure were more extensively characterized using human Ads from serotypes 2 and 5 [1]. As a result, the majority of adenoviral vectors (AdV) were derived from these serotypes. Following receptor interactions, the virus is internalized by means of clathrindependent endocytosis and is rapidly transported to the nucleus. The Ad2 genome is shown schematically in Fig. 1A. It contains cis-acting elements at each end, the inverted terminal repeats (ITRs), which are involved in DNA replication and packaging (at the left ITR). Viral genes are expressed in two phases delineated by the onset of DNA replication; the early phase, which lasts about 6–8 h, and the late phase, which culminates around 30 h post-infection with the production of more than 104 infectious virions per cell. Genes from the early phase are subdivided into six transcription units: E1A, E1B, E2A, E2B, E3, and E4. Each region gives rise to a set of alternatively spliced transcripts from which several proteins are synthesized, whereas two delayed early units produce only one protein, respectively, pIX and IV2a. The roles of the early gene products can be generalized as follows. The E1 genes are involved essentially in activating expression and diverting the cellular machinery toward viral DNA replication and preventing apoptosis. The E2 region bears genes directly involved in viral DNA replication. The genes from E3 region are involved in hampering the host defense system. Finally, the six ORFs forming the E4 region are involved in viral mRNA processing and transport as well as regulation of transcription and apoptosis. Although the function of some of these gene products is unknown, most of them are multifunctional while many share complex interactions with each other. Late genes are expressed from five families of transcripts (L1–5) and encode proteins involved in encapsidation and maturation of viral particles (reviewed in [2]).
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Fig. 1. (A) Simplified schema of the transcriptional map of Ad2/5 (36 kb). Arrows represent viral transcripts, shaded arrows represent transcriptional units. E, early expression; L, late expression. The ITRs and the packaging signal ( ) are indicated. Light-shaded portions of the genome represent DNA that was deleted in several recombinant AdV. (B) Schema of the adenovirus vectors currently reported in the literature. Deletions characterizing the first, second, third generations, and replicative AdV are presented as follows, in order of appearance. 1st generation: E1E3. 2nd generation: (E1, E2B, pTP, E3), (E1, E2A, E3), (E1, E3, E4), (E1, E2A, E3, E4). 3rd generation: all genes deleted, all genes deleted but pTP.
111 In the last two decades Ads have been extensively used for gene transfer into mammalian cells (reviewed in [3]). Several features make AdV attractive vehicles for gene transfer and protein production. First, AdV can easily be produced at high titers; second, they can transduce a large variety of cell types; third, they do not require cell division for transgene expression; and fourth, their genome remains episomal, therefore they do not permanently alter the target cell. AdVs have been extensively engineered in order to be adapted to several applications of gene transfer in mammalian cells and gene therapy in vivo. In this chapter, we aim to provide the reader with an overview of the AdV platforms currently developed, with means to engineer, produce, and characterize them.
2. Adenoviral vectors To effect the transfer of exogenous genes into mammalian cells, the Ad has been modified in order to: (1) eliminate its replication capacity; (2) increase its cloning size capacity; (3) reduce the host immune response; and (4) retarget it to specific cell receptors. The most widely used AdV is the so-called first generation that is deleted in the E1 and the E3 regions. Later, other modifications were made in order to meet specific objectives. The various AdV engineered to date are depicted in Fig. 1B. 2.1. First generation AdV A deletion that removed the E1A and E1B genes distinguishes the first generation AdV from the wild type Ad. The E1 deletion renders the AdV replication dependent on cell lines such as the 293, A549-E1, or PER.C6, which complement the E1 functions [4]. The E3 region is not required for viral production in vitro and thus its deletion increases the cloning capacity without having to complement its function in producing cells. Those vectors can accommodate exogenous DNA of up to 8 kb in size. Construction of first generation AdV is routinely performed by recombination between a digested AdV genome and a transfer plasmid that contains the left arm portion of the AdV, the transgene cassette, and a portion of homologous sequence for recombination to occur. Recombination is performed in mammalian cells, generally in 293 cells, which are readily transfected by the calcium phosphate procedure. The recombination requires the co-transfection of both the transfer plasmid and the viral genome. This procedure, although favored for its simplicity, is inefficient. Recombination is a rare event and incompletely digested AdV may give rise to non-recombinant AdV. Our laboratory is relying on co-expression Replicative AdV: PS, 100K, E1B-55K, E1A promoter substitution. Abbreviations: ITR, inverted terminal repeat; pol, Ad polymerase; pTP, preterminal protein; PS, Ad protease; DBP, DNA-binding protein; E1, E2, E3, E4, early phase transcriptional units; L1, L2, L3, L4, L5, late phase transcriptional units.
112 of the green fluorescent protein (GFP) to select recombinant plaques under the microscope [5]. Several of our transfer vectors carry a cassette, which joins the GFP with the transgenes through an internal ribosome entry site (IRES). In this way, GFP expression is linked to transgene expression, and recombinant plaques can be distinguished from non-recombinant ones. Other strategies have been developed in order to minimize contamination with non-recombinant AdV and to reduce the time required for AdV production. These strategies rely on a viral genome that is carried as a plasmid or cosmid, and recombinant AdV can be produced by several means: (i) subcloning directly the transgene into the plasmid containing the viral genome; (ii) using a recombinase such as Cre or Flp that facilitates specific and efficient introduction of transgenes into the viral genome; or (iii) using bacterial recombination for introduction of the transgene into the viral genome. Strategies of direct cloning into AdV genome serve the purpose of eliminating non-recombinant plaques, therefore plaque purification. Cloning in large plasmids is difficult; thus cosmids were designed to facilitate the subcloning steps [6]. The strategy making use of recombinases replaces the subcloning step through efficient homologous recombination either in bacteria or in mammalian cells. Such strategies require AdV that are engineered to contain recombination sequences, such as loxP or frt, which are substrates for Cre and FLp, respectively, and cell lines or bacteria expressing the specific recombinase [6,7]. Recently, a novel strategy based on positive selection was developed for producing recombinant AdV. The strategy consists of inserting the Ad protease gene in the shuttle plasmid, to complement a protease (PS) deletion in the virus backbone used for recombination. Since the virus backbone cannot form viral particles in the absence of the PS, the only infectious particles produced are those that have recombined with the complementing shuttle plasmid. Thus, following transfection of permissive cells with the shuttle plasmid and infection with an AdV deleted for its PS gene, pure recombinant plaques are produced within 2 weeks with an efficiency of 100%. For the construction of recombinants one at a time, this positive selection method is at least as efficient as the best methods developed so far. Furthermore, this represents the first method allowing for easy construction of AdV libraries with high diversities [8]. 2.2. Second generation AdV First generation AdV can sustain transgene expression for years in post-mitotic cells of immunodeficient animals. However, their expression will persist only a few weeks in actively growing cells, due to the dilution of the viral genome by cell division, or in immunocompetent animals, due to the host immune system that destroys the transduced cells. Despite the absence of E1 genes required to initiate expression of viral genes, de novo viral gene expression occurs to some extent. Viral proteins are then exposed to the cell surface and elicit an immune response. In order to minimize this unwanted expression, AdV have been further crippled by deletion of genes in the E2 or E4 regions. The production of these second generation AdV has required the
113 development of complementation cell lines expressing the corresponding deleted genes. Deletions were engineered in E2A, E2B, DNA polymerase and/or preterminal protein (pTP), as well as E4, and in most cases in vivo studies showed significant reduction of the immune response. In the case of E2B, the deletion of either the polymerase or pTP alone decreased hepatic toxicity and prolonged vector stability, while combining both deletions resulted in further improvement [9]. In the case of E4 deletions, the generation of Ad E1 E2A E4 demonstrated that further crippling of AdV by removing E4 genes reduced vector toxicity but also reduced vector stability [10]. In fact, it appears that ORF3 is required for efficient expression of transgene under the control of several promoters used in AdV, particularly the CMV promoter [11,12]. The mechanism responsible for the reduced expression and stability of second generation AdV deleted in E4 remains to be elucidated. Nonetheless, most studies using E4-deleted AdV have reported decreased transgene stability, unless the ORF3 or ORF6 have been reintroduced. Moreover, reintroducing ORF3 in E4-deleted AdV restores its stability while retaining a reduced toxicity. On the other hand, ORF6 reintroduction restores both stability and hepatotoxicity [13]. It is noteworthy that either ORF3 or ORF6 can complement the complete E4 deletion. 2.3. Third generation AdV Because second generation AdV were still sub-optimal in escaping the immune response, and there was a need for larger exogenous DNA insert capacity, the more drastic approach of eliminating all viral genes was undertaken. Fully deleted vectors have been given several names: third generation, helper-dependent, high capacity, or gutless. These AdV offer three main advantages: (1) no expression of viral genes is possible, thus reducing the immune response; (2) a cloning capacity of up to 36 kb; and (3) they retain all of the advantages of first generation AdV with respect to transduction efficiency. However, they are more difficult to produce since it is almost impossible to generate a stable cell line that would complement all of the Ad genes, due to the toxicity associated with the over-expression of most of the Ad proteins. The helper functions required for the complementation of third generation AdV are most often provided by co-infection with a helper virus, but they can also be provided by co-transfection of the adenoviral genome as plasmid [14], or through a baculovirus/Ad hybrid [14–16]. The dependency on a helper Ad results in decreased viral yields and contamination with helper viruses. A few strategies have been designed to overcome these drawbacks. The strategy that is currently favored is to remove the helper virus-packaging signal through Cre/loxP recombinase [17], and more recently the Flp/frt recombinase system [18]. These systems require that third generation AdV be amplified with a helper Ad bearing loxP or frt sequences flanking its packaging signal. In 293 cells expressing Cre or Flp recombinases, the packaging signal of the helper virus is deleted resulting in viral DNA that still can produce viral proteins, but cannot be packaged into viral particles. Although the cleavage by Cre or FLP is not 100% and some helper viruses are still packaged, this procedure
114 reduces contamination to less than 0.01% after CsCl purification in the best stocks. While helper AdV are a very small proportion of the preparation, high-dose injections such as 1012 particles would still deliver more than 108 contaminating particles. This might be sufficient to elicit an immune response. Therefore, further improvement in third generation AdV purity is required. Another issue about the production of third generation AdV is the requirement of an optimal genome size of 26–38 kb for efficient particle formation. In order to generate optimized third generation AdV, the large amount of deleted DNA must be replaced with stuffer DNA that is carefully chosen. Thus, the stuffer DNA should have the following desirable characteristics: (1) it should be preferentially of mammalian origin; (2) it should be free of repeats, retrovirus elements, and genes; (3) contiguous human DNA should be fragmented to prevent integration; and (4) a matrix attachment region might confer stability [19]. Other cis-acting elements, such as a 400-bp fragment from the right end of Ad, which appears to confer growth advantage, can contribute to improved yields of gutless AdV [19]. While efforts are still ongoing to further optimize their production, several studies performed with third generation AdV have provided very promising results in reducing immune response and prolonging stability. The hurdles remain essentially in the production of clinical grade third generation viruses due to the complexity of the process. 2.4. Replicative AdV While a major concern in the use of AdV for gene replacement therapy is reduction of toxicity and host immune response, there are applications, such as vaccination or cancer gene therapy, where toxicity and immunogenicity are rather an asset. For such applications the increased transgene expression through an increase in copy number following viral DNA replication can also be advantageous. This can be done by deleting genes that do not affect DNA replication, such as the PS or the 100K (late genes), while interfering with the production of infectious particles [20,21]. Although the full potential of such AdV has yet to be demonstrated, they could have applications for recombinant protein production, vaccination, cancer gene therapy, and production of adeno-associated viruses (AAV). Other types of replicative AdV were developed more specifically for cancer gene therapy. Because transduction efficiency of 100% is not achievable, yet complete eradication of tumor cells is required for successful treatment; the ability of transduced tumor cells to kill bystander tumor cells is of paramount importance. One approach to achieving this goal is to engineer AdV that can replicate and form mature particles only in tumor cells. This was achieved using AdV deleted either in the E1B-55 kDa and/or the E1B-19 kDa gene. Thus, after infecting a few tumor cells, the progeny of conditional replicative oncolytic AdV can spread and destroy neighboring tumor cells while sparing normal cells. Other strategies rely on a tumorspecific promoter to drive E1A gene expression [22]. The above strategies have also been combined with cytokine or suicide genes to further improve the efficacy of conditional replicative AdV.
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3. Gene delivery 3.1. Cell entry pathway Effective vectors are those which deliver the transgene specifically to the appropriate tissues. Strategies for targeting AdV to specific cell types became possible following the improved understanding of the viral entry mechanisms, including the identification of the viral proteins and cellular receptors that are involved in virus–cell interactions. 3.1.1 Ad capsid proteins involved in virus entry Two capsid proteins, the fiber and the penton base, play essential roles in virus–cell interaction and virus internalization. Five penton base subunits are associated with each fiber, itself composed of a homotrimer of fiber proteins. The fiber is responsible for the assembly and stabilization of the virion. It can be divided into three domains with distinct functions: (1) the short amino-terminal tail is involved in its association with the penton base protein through an FNPVYP motif. This interaction anchors the fiber to the Ad capsid. Also located in this domain is a nuclear localization signal (KRXR), which directs the intracellular trafficking of newly synthesized fibers to the cell nucleus where Ad particles are assembled. (2) The central shaft domain, whose length varies among different serotypes, is characterized by sequence repeats of approximately 15 residues. The shaft domain, which makes the carboxy terminal end of the fiber extending away from the virion, therefore provides optimal condition for fiber–receptor recognition. (3) The carboxy terminal knob domain forms an sandwich structure, which contains two anti-parallel -sheets named R and V. These sheets are each composed of four strands linked by loops in which several residues are involved in the binding of the virus to the coxsackie virus and Ad receptor (CAR). Another distinct function carried out by the knob domain is the initiation of fiber trimerization. The penton base subunit contains a flexible loop structure consisting of two stretches of helices. The loop of the five penton base units forms a protrusion on the protein surface, exposing an arginine–glycine–aspartic acid (RGD) motif (aa 340–342), which interacts with the cellular integrin v, and a leucine–aspartic acid–valine (LDV) sequence (aa 287–289) also identified as an integrin-binding motif. In addition, the penton base plays an important role in the internalization of viral particles and release of capsids from the endosome. 3.1.2 Cell receptors CAR, a 46-kDa transmembrane glycoprotein widely expressed in different tissues, is the primary receptor for Ad transduction. It belongs to the immunoglobulin (Ig) superfamily and contains two extracellular immunoglobulin-like domains, a hydrophobic transmembrane region and an intracellular cytoplasmic domain. Functional analyses of recombinant forms of CAR have demonstrated that the first immunoglobulin-like domain is sufficient for Ad binding. It should be noted that certain subgroup B or D Ads do not use CAR as receptor for their entry. Also, an
116 sialic acid residue of some glycoproteins is used by Ad37 as receptor for their cell entry [23]. Interaction of the fiber with CAR leads to virus attachment onto the cell surface, but, in general, this is not sufficient for rapid virus uptake. It is the association of the cellular integrins v3 or v5 with the viral penton base that facilitates virus internalization. Moreover, fiber-less particles have been demonstrated to keep their infectious capacity for monocytic cells via an integrin-dependent pathway [24]. Recent studies have suggested that heparan sulfate glycosaminoglycans are sufficient to mediate Ad2/5 binding on the cell surface and infection [25]. The 2 domain of the major histocompatibility complex class I protein (MHC-I) is also reported to function as an alternative receptor [26]. However, when CAR and MHC-I proteins were co-expressed on the same cell, only CAR had high affinity for the Ad5 fiber. MHC-I-dependent Ad5 attachment may only occur when CAR expression is low. 3.1.3 Mechanisms of early steps of cell infection by Ad Cellular uptake of the Ad particle is a two-step process: (1) an initial interaction of the fiber with cellular receptors like CAR or MHC-I [27]; and (2) the internalization of the virus mediated by the interaction of the RGD motif of the penton base with other cellular receptors, such as the integrins v3 and v5. This latter interaction triggers the phosphorylation of several signaling molecules, and induces a signaling cascade that promotes actin polymerization, which provides the mechanical force necessary for the internalization of clathrin-coated pit containing AdV. Viral proteins begin to be dismantled during the endocytotic uptake from the plasma membrane and during subsequent changes in the endosomes. After the endosomal membrane is lysed, the uncoated particle escapes to the cytoplasm with the help of the penton base protein, and the core is translocated to the nucleus through nuclear pores. The Ad genome then enters the nucleoplasm where viral replication takes place. 3.2. Targeting of AdV Active research is ongoing to develop targeting vectors to selectively transduce the tissue of interest for the following reasons: the Ad wide tropism leads to indiscriminate transduction of both target and bystander cells. Transduction of nontarget cells not only dilutes the amount of therapeutic viruses delivered to target cells but also results in undesirable transgene expression. Moreover, because some cells have no or low levels of CAR, targeting AdV to a specific cellular receptor would overcome the problem of poor transduction efficiency. Finally, preventing AdV interaction with antigen-presenting cells should reduce the humoral and cytotoxic T lymphocyte response caused by AdV in vivo. Since the fibers of different Ad serotypes display various amino acid sequences, it was suggested that they might recognize different receptors and consequently have different tropism. Therefore, several studies were undertaken using either chimeric
117 fibers or exchanging fibers from different serotypes in order to alter AdV tropism. For example, Shayakhmetov et al. [28] demonstrated that the chimeric vector Ad5/ F35 can successfully infect human CD34 þ cells by CAR- and v integrinindependent pathways. However, effective targeting requires the abolition of vector interaction with its natural receptors, and the redirection of the vector to another type of receptor, which is specific to the target cells. Two strategies have been developed to realize this goal: (1) a conjugate system that allows AdV interaction with bridging molecules for the binding to a new receptor; and (2) a genetic approach in which the virus capsid proteins are modified to recognize only the target receptor (Fig. 2A).
Fig. 2. (A) Schema depicting two strategies to retarget AdV. Left, the natural tropism of AdV and its inability to interact with certain target cells. Middle, the use of a bifunctional molecule bridging the virus to the target cell. Right, the genetic modification of AdV as the alternate strategy. (B) Special packaging cells need to be engineered for production of genetically modified AdV that may fail to infect ‘‘normal’’ packaging cells.
118 3.2.1 Conjugate targeting system This system makes use of a bispecific molecule, which binds to the AdV and to the targeted receptor. The AdV specificity is usually provided by a soluble form of CAR, the binding domain of MHC, or neutralizing antibodies against one of the capsid protein. Other conjugation strategies include biotinylation, and coating with polyethyleneglycol (PEGylation) or a multivalent hydrophilic polymer. The cellular specificity can be generated with an antibody or a high-affinity ligand specific for a receptor or an adhesion molecule expressed on the targeted cell surface, such as CD40, v integrin, the receptors for fibroblast growth factor (FGF), tumor necrosis factor (TNF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and several tumor markers. The bispecific molecules were made by chemically linking two molecules, or by engineering recombinant fusion proteins. Any receptor can be targeted with the above strategies because any high-affinity antibody can be used as ligand. An additional advantage of the PEGylation and multivalent polymeric modification of AdV is that vectors are protected against neutralizing antibodies, thus avoiding some of the immune responses. However, some limitations remain to be overcome: there are several steps involved in the largescale production and purification of AdV and bispecific molecules in the development of stable forms of the complexes for storage and for intravenous administration. Thus, the development of other approaches, such as genetic retargeting, may be more promising. 3.2.2 Genetic targeting system This approach relies on genetic modifications to incorporate targeting ligands to the coat proteins of AdV. One challenge is to determine which locations are appropriate for incorporating high-affinity ligands. Wickham et al. [29] have constructed AdV whose fibers contain an RGD motif or a polylysine in its C-terminus. These viruses increased transduction in multiple cell types lacking high levels of CAR. The HI loop of fiber, the RGD loop of penton base, and an exposed loop of hexon were also chosen for the insertion of high-affinity peptides. Modifications in these locations were demonstrated to lead to virus attachment and cell entry in a CAR-independent manner. Another issue concerns the size of the peptide to be inserted. The incorporation of a ligand into Ad capsid proteins must not alter the conformation of these proteins, and should not adversely affect their other normal functions. Ideal ligands are small peptides specific for the targeted cellular receptor. Published findings suggest that less than 25–30 residues can be inserted into the C-terminus of the fiber, because longer sequences destabilize the fiber trimer [30]. It is also reported that ligands added into the HI loop of the fiber should not exceed 63 amino acids [27]. Full re-targeting requires the elimination of AdV interaction with its natural receptors. Several amino acid residues localized in the knob region of Ad5 fiber, such as S408, P409, K417, K420, K442, Y477, L485, Y491, A494 and A503 are directly involved in fiber–CAR interaction. Mutations of these amino acids and of residues 489–492 lead to a drastic reduction in virus transduction [31]. Several studies have successfully combined CAR-binding ablation and targeting ligand insertion for retargeting. For example, Nicklin et al. [32] have retargeted AdV to endothelial
119 cells by mutating two amino acid residues (S408E and P409A) in the knob region that blocks CAR interaction, and by incorporating the SIGYPLI peptide, which was demonstrated to bind specifically to endothelial cells. Finally, disruption of v integrin at the same time is also critical for fully ablating the natural AdV tropism. The production of a fully ablated AdV in its endogenous receptor interaction requires: (1) the insertion in the Ad capsid protein of a novel ligand unable to bind to any cells; and (2) the use of novel packaging cells expressing a chimeric receptor that allows entry and replication of the modified AdV [33,34]. Therefore, in addition to the targeting ligand incorporated into the AdV capsid for cell-specific transduction, another pseudoreceptor-binding ligand should also be inserted into the vector for entry into the packaging cells (Fig. 2B). This pair of pseudoreceptor–ligand for virus propagation should preferably be completely artificial, otherwise it might compromise the specificity of the retargeting, since any natural analogue could cause binding of the vector to non-target cells in vivo.
4. Gene expression 4.1. High-level expression Besides transduction efficiency, transgene expression efficiency has also been improved. High expression level of recombinant proteins with low AdV multiplicies of infection (MOI) are desirable to minimize negative effects of high MOI on target cells. Strong promoter-enhancer elements usually derived from viruses have been used to construct expression cassettes. For example, the immediate-early human cytomegalovirus (hCMV) promoter has been utilized extensively because of its high transcriptional activity in a wide range of cells. However, AdV containing standard CMV-based expression cassettes produce proteins at suboptimal levels representing no more than 2–3% of total cell protein (TCP). Optimizing the expression cassette by including the Ad tripartite leader and a small intron as in pAdCMV5 improved performance by 6–12-fold, both at low and high MOI, and achieved up to 35% of TCP in AdV-infected cells at high copy number [5,35]. The expression of some proteins may have deleterious effects on the host cell or on Ad production. Inducible gene expression systems (Chapter 22) have been therefore developed to overcome this problem. Regulation of transgene expression in vivo can also improve its safety and efficacy. An ideal inducible system would have low basal expression of transgene without the inducer and very high expression in its presence (reviewed in [36]). The most widely used inducible promoter is the tetracyclinecontrollable transactivator system. A trans-acting factor (tTA) is formed by the fusion of the activation domain of the herpes simplex virus (HSV) protein VP16 with a tetracycline repressor protein from Escherichia coli. The tTA transactivator stimulates transcription from a promoter containing the tetracycline operator sequences (tetO), but the interaction of tTA with tetO is prevented in the presence of tetracycline at concentrations not toxic to eukaryotic cells. The use of an optimized tet-regulated promoter, as in pAdTR5, allows the expression of proteins that are
120 cytotoxic or interfere with Ad replication, at 10–15% TCP levels [5,35]. Recently, a new inducible gene switch was developed in our laboratory using the operator sequences and repressor of a bacterial p-cymeme operon whose DNA-binding can be regulated by cumate. This cumate-inducible system is comparable to the tet system in many respects. Interestingly, the cumate-inducible promoter achieved higher transgene expression than the tet-regulated promoter at lower MOI, thereby minimizing the interference of AdV at higher MOI. Moreover, combining the tet and cumate regulatory elements reduced leakiness while fully maintaining the expression level of the on state. Several other inducible systems have also been tested in AdV. These include the cre-loxP system from bacteriophage P1, the bacteriophage T7 binary system [37], the insect ecdysone system and the human rapamycine-inducible system. These inducible systems have intrinsic advantages and limitations that should be carefully evaluated in comparative studies in order to select the optimal system for a specific application (reviewed in [38]). 4.2. Specific transgene expression Many studies have explored the use of tissue-specific promoters mainly to restrict transgene expression to the target tissues in order to avoid unwanted expression and reduce the immune response against the transgene product. Tissue-specific promoters are also used to transduce particular types of tumor cells in cancer therapy, as discussed above. 4.2.1 Tissue-specific promoters Tissue-specific targeted gene transcription was mainly attempted for expression in the lung, epithelia, liver, pancreas, muscles, neural cells, mammary gland and cardiac cells. For example, the amylase promoter is used to target pancreas cells, truncated muscle creatine kinase (MCK) and the smooth muscle alpha-actin (SMA) promoters for targeting skeletal and smooth muscle cells, ventricle-specific myosin light chain 2, ventricle specific -myosin heavy chain and troponin T promoters are used for cardiac cells, L7/PCP2 promotor for cerebellar Purkinje cells, and synapsin 1, tubulin alpha 1 and neuron-specific enolase (NSE) promoters are used to target neurons. Using silencer elements is another approach for specific transgene expression. For example, elements specific to neurons can selectively repress the transcription of genes in non-neuronal cells so that when they are cloned upstream of the ubiquitous phosphoglycerate kinase promoter, transgene expression is restricted to neurons [39]. 4.2.2 Tumor-specific promoters Transcriptional targeting of tumors has been investigated with promoters known to be particularly active in tumor cells. The use of tumor-specific promoters should result in expression of a therapeutic gene primarily in tumor cells, while marginal expression should be observed in normal cells. There are several promoters that meet this objective: the MUC1/Df3 promoter has been used for breast carcinoma, the alpha fetoprotein (AFP) promoter for hepatomas, the cyclooxygenase-2 (cox-2)
121 promoter for gastrointestinal cancers, the CTP1 promoter for colon cancer, the prostate-specific antigen (PSA), human glandular kallikrein (hk2) and probasin promoters for prostate carcinoma, and the Midkine (MK) promoter for neuroblastoma. Promoters that are particularly efficient in actively dividing cells are also useful for cancer targeting, since in adults, few cells other than tumors are replicating. The E2F-responsive promoter E2F-1, is one of them. It allows eradication of established gliomas with significantly less toxicity to normal tissue than constitutive promoters [3]. A similar approach made use of the hexokinase type II promoter to preferentially drive the expression of a toxic gene in a variety of cancer cell lines [3]. The human telomerase reverse transcriptase (hTERT) promoter was also shown to be strong and preferentially active in tumor cells. This promoter enabled induction of the Bax gene to elicit tumor-specific apoptosis in vitro, and in vivo it caused the suppression of tumor growth in nude mice while preventing the cytotoxic effects of the Bax gene to normal cells [40].
5. Production and analyses of viral particles First-generation AdV are commonly produced in 293 cells that express the E1 genes from a large fragment of the left end of the Ad genome integrated in their chromosomal DNA. However, homologous sequences between AdV and the genome of 293 cells allow for occasional recombination leading to the generation of replication-competent AdV (RCA). This problem has been solved by the development of new E1-complementing cell lines, which do not share homologies with E1-deleted AdV [3,4]. Pharmaceutical and commercial applications of AdV require virus production on a large-scale. This has been facilitated by the development of processes allowing the production of AdV in suspension and in serum-free cultures [41]. Following production, AdV lysates are prepared by three freeze-thaw cycles and removal of cellular debris. These viral crude lysates can readily be used for in vitro studies. However, for many applications it is preferable to purify and concentrate the AdV particles. This is most often done by CsCl gradient purification, which relies on the specific buoyancy of AdV. However, this procedure is time-consuming and difficult to scale-up, necessitating the development of other purification protocols based on chromatography [42]. For the production of clinical-grade material, several challenges remain to be addressed to make the process economically viable and fully compliant with regulatory requirements. Thus, the yield, homogeneity, lot consistency, and a reliable characterization of the vector particles and infectivity need to be addressed. Regarding consistency and comparability of AdV production among different laboratories, the establishment of a standard sample and of standard operating procedures (SOPs) was awaited. The Adenovirus Reference Material Working Group (ARMWG) has established such standards. A wild-type Ad5 will be available from the ATCC in 2002, and SOPs for evaluation of viral particles and infectious particles are published at http://www.wilbio.com. The establishment of this standard
122 is crucial to the comparability of AdV quantification across different laboratories. Current titration measures have different efficiency and are all extremely sensitive to absorption conditions (time, volume, agitation). Moreover, the intrinsic sensitivity of each assay varies within one order of magnitude (TCID50, plaque assay, transduction units). A report on the AdV standards has been published [43]. Finally, AdV need to be stored under conditions that ensure their stability over time. Croyle et al. [44] have developed a formulation for the long-term storage of lyophilized AdV at room temperature without loss of virus stability. Recently, the ARMWG has proposed a rather simple formulation that performed well for a period of over 6 months. This formulation is composed of 20 mM Tris pH 8.0, 25 mM NaCl, 2.5% glycerol, and is suitable for the stable storage of AdV at a concentration below 5 1011 particles [45].
6. Conclusion The biological features of AdV have contributed to their popularity as tools for functional studies and gene therapy applications as demonstrated by the thousands of reports on AdV in recent years. Because efficacy and specificity is of paramount importance in gene therapy success, considerable efforts have been invested on their improvement. Although several challenges in targeting and reducing the host immune response remain to be addressed, current progress has armed molecular biologists with a wealth of AdV adapted to numerous applications. Moreover, new developments in the construction of Ad-retro, Ad-AAV or Ad-EBV hybrid viruses will further expand their use in therapy that requires long-term transgene expression. Finally, the recent development of Ad-based libraries [8] will lead to new applications yet to be explored.
Acknowledgements We thank Re´nald Gilbert for critical reading of the manuscript. This is a NRC publication #37701.
Abbreviations AAV Ad AdV ARMWG CAR CMV CPE GFP
adeno associated virus adenovirus adenovirus vector(s) adenovirus reference material working group coxsackie virus and adenovirus receptor cytomegalovirus cytopathic effect green fluorescent protein
123 IRES ITR MHC MOI ORF pTP PS RCA SOP TCP tetO tTA
internal ribosome entry site inverted terminal repeats major histocompatibility complex multiplicity of infection open reading frame preterminal protein adenovirus protease replication-competent adenovirus standard operating procedure total cell protein tetracycline operator tetracycline trans-activator
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