A number of hybrid adenoviral vector systems have been reported in the literature, combining the properties of RV, AAV, and EBV vectors, as well as elements of other Ad serotypes, to enhance the therapeutic efficacy of Ad vectors in vivo. The principal aim of these new hybrid vectors is to overcome the limitations of transient Ad vector retention in infected cells. In addition to the well-documented limitations of Ad vectors (Table II), some initially perceived advantageous properties of Ad vectors do actually limit their effectiveness toward therapy for some diseases. The broad host range of Ad vectors induces significant disadvantages when tissue targeting is required and compromises systemic administration. Additionally, the low pathogenicity of adenoviruses in humans has resulted in many serotypes, including the conventional vector strains of Ad2 and Ad5, being endemic. Hence a potent natural anti-adenoviral immunity is fashioned generally at a very early age. The highly immunogenic nature of the proteinous Ad virion further confounds the system, with a rapid and highly effective host humoral response being developed to the Ad vector. Research is thus being channelled into both retargeting Ad vectors to specific tissues and silencing the structural immune stimuli to facilitate enhanced Ad vector transduction.
As targeting and humoral immunity are connected in essence to the same surface moieties of the Ad particles, both disciplines are fundamentally interlinked. Methods applied to limit the humoral responses have focused on two main strategies: application of alternative "immune silent" Ad serotypes or display of alternative ligands on the surface of the virions, which is also the major strategy for retargeting the vector.
The use of alternative serotypes enables the consecutive application of immunologically distinct Ad particles, enabling avoidance of specific humoral responses to previously applied vectors [54, 55]. This system has presented some success in vivo , although the presence of cross-reacting antibodies is problematic due to the evolutionary similarities of Ad serotypes. The application of alternative Ad serotypes with different surface markers also provides a mechanism of alternative targeting, as different serotypes possess tropism for different tissues in the human body. For instance, the conventional gene therapy subtypes Ad2 and Ad5 have natural tropism for the gut epithelial layer. Hence, in terms of gene therapy for cystic fibrosis, initial vectors proved disappointing due to their low infectivity of the airway epithelia. To overcome this restriction, Zabner and colleagues investigated other Ad serotypes for airway epithelia tropism , A number of other Ad serotypes, specifically Ad 17, were found to infect the airway epithelia with increased efficiency to wtAd2 . They therefore proceeded to generate Ad2 hybrid vectors pseudotyped with the Adl7 fiber, where the endogenous Ad2 fiber gene was replaced with the Adl7 fiber gene. The resultant chimeric vector displayed increased efficiency of binding and gene transfer to well differentiated human epithelial cells. A similar study by Croyle and colleagues demonstrated that wild-type Ad41 had enhanced transduction properties in intestines compared to Ad5 . These studies emphasize the potential of alternative Ad serotypes with tropism for different tissues in the human body. Pseudotyping also provides an invaluable mechanism of integrating alternative serotype fiber (and/or penton base) genes from other Ad serotypes into the currently well-researched Ad vectors, without having to reconstruct the vector backbones. The use of nonhuman adenoviruses as vectors for gene therapy is also under investigation, with bovine, ovine, canine, feline, and avian adenoviruses being researched [59-62]. As well as being potentially unexposed to the immune system, they may also have specific tropism for selective tissues in humans. The potential of pseudotyping nonhuman Ad vector components with conventional human Ad vectors is therefore of interest.
The use of targeted viral vectors to localize gene therapy to specific cell types introduces significant advances over vectors with conventional natural tropism. As well as the safety aspects of reduced immunogenicity and toxicity, the reduced uptake by nontargeted cell types may enable application of systemic delivery with feasible viral titers and loads. In order to retarget Ad vectors, first the natural tropism of the virus must be removed and, second, novel, tissue-specific ligands introduced . Two main mechanisms have been used to retarget Ad vectors. First, the use of external molecules with affinities for both the Ad surface structural moieties as well as a cell-type-specific surface ligand. These bispecific molecules act as bridges between the virions and the cell. A neutralizing antibody or high-affinity peptide for the fiber or penton base can act as the Ad-binding moiety, which can be covalently linked to a high-affinity ligand for a tissue-specific receptor . A drawback of the bridging molecule approach is that native receptor binding is never 100% blocked. To truly block native Ad binding to its cognitive receptor, removal of the intrinsic receptor binding domains is required.
A second approach involves creation of hybrid Ad vectors, pseudotyped with novel receptor recognition functions. Genetic modification of the Ad genome by incorporating targeting ligands inside the genome, while deleting or ablating sequences of the penton and fiber involved in receptor recognition, has been reported. High-affinity peptide motifs have been subsequently demonstrated to be functionally incorporated into Ad particles. These "proof-of-concept" studies focused on the incorporation of ligands without ablating natural receptor interactions and resulted in expanding the vector tropism, which has proved beneficial in vivo in transducing both vascular smooth muscle and some tumor types [64-66]. Future studies will focus on honing the targeting functions to specific cell types. High-affinity ligands have been stably inserted into the HI loop or on the C-terminus of the fiber or into the integrin-binding RGD domain of the penton base . However, the size, location, and type of ligand to be inserted are currently under debate and remain to be determined. Wickham and colleagues demonstrated 10- to 1000-fold reductions in transduction of cells expressing the coxsackievirus and adenovirus receptor (CAR) with CAR-ablated vectors , the residual transduction being pentonbase-mediated, emphasizing the requirement for additional ablation of penton base binding . The further requirement of novel packaging cell lines to facilitate infection and propagation of the CAR/integrin-binding ablated particles also remains an issue.
B. Adenoviral/Retroviral Hybrid Vector Technologies
A hybrid vector system incorporating the advantageous long-term stable integrative functions of retroviral vectors into adenoviral vectors could provide a major clinical advancement to gene therapy. Hybrid vector systems are thus being investigated, incorporating retroviral components into the backbones of adenoviral vectors. Initial studies have focused on utilizing adenoviral vectors as directors of retroviral vector production, delivering the gag, pol, and env genes as well as retroviral LTR cassettes to cell populations both in vitro and in vivo.
Conventional retroviral packaging cell lines are stably transformed with gag, pol, and env functions and release retroviral particles upon plasmid transfection of a retroviral LTR transgene cassette . High-titer retroviral stocks of greater than 107 infectious units (iu)/mL can now be obtained from conventional stable producer cell lines . To achieve the highest vector titer, it is necessary to select clones of vector-transduced cells individually due to the varying titers of producer cell clones . Direct injection of retroviral vectors in vivo has, however, yielded limited efficiencies due to the limited transducing titers and poor infectivity. Application of retroviral vectors in the clinic has thus focused on ex vivo protocols. This involves the removal of patient tissues, which can be cultured for a brief period in the laboratory, transduction with the RV vector, and reimplantation back into the patient. The ex vivo approach has yielded some success, although the procedure is cumbersome and costly, and in most cases, it can only transduce a small fraction of the target cells [68, 69]. The establishment of retroviral producer cells in situ provides a further mechanism of enhancing the efficacy of retroviral gene therapy. Transient transfection of target cells in vivo with the retroviral vector and packaging plasmids, previously used to generate producer cell lines in vitro, by direct DNA injection has been reported , Although stable integration of subsequently generated retroviral particle genomes could be detected, the efficiency was very low. The implantation of retroviral producer cell lines into patients has presented a far greater potential for the in situ production of retroviral vectors. Gene therapy using MoMuLV-based producer cells to treat brain tumors  has been carried out in a clinical trial, but no clear clinical benefit has been reported to date.
The infectivity of Ad vectors both in vitro and in vivo provides great potential in increasing the efficiencies of retroviral production technology. The group of David Curiel pioneered the development of hybrid retroviral/adenoviral vectors by using the infectivity of adenoviral vectors to efficiently deliver the requisite retroviral packaging and vector functions to target cells in vivo, thereby rendering them retroviral producer cells in situ (Fig. 2). The subsequent release of high local concentrations of retroviral particles in situ would enable stable transduction of neighboring tissues, for the transient period of adenovirus transduction. The Ad/RV hybrid system reported by Feng and colleagues utilized a two-adenovirus delivery strategy . The first adenovirus contained an LTR-flanked retroviral vector cassette encompassing the GFP marker and neomycin resistance genes: Ad/RV-vector. The second adenovirus contained the replication-defective retroviral helper machinery, carrying the gag, pol, and env genes of MoMuLV: Ad-gag/pol/env. High-titer adenoviral vectors could be generated containing the RV cassettes, which could efficiently direct the in vitro packaging of RV particles at titers similar to conventional packaging cell lines [72, 67]. These studies clearly demonstrate the compatibility of both the adenoviral and the retroviral life cycles in the context of a hybrid vector configuration.
Upon infection of cells in vitro with the Ad/RV vector alone, high initial levels of GFP expression were observed but gradual loss of expression was documented over a period of 60 days as the nonintegrated adenovirus was lost from dividing target cells. Conversely, upon application of both adenoviruses to cells in vitro, GFP expression was persistent for extended periods of time. However, the persistent level of gene expression was reduced beyond the time at which expression could be solely attributed to the Ad/RV vector. The stable integration of the retroviral cassette in surrounding cells was believed to be responsible for this extended expression. The longer term GFP-expressing cells in cultures transduced with both Ad vectors, were present in clustered outgrowths, suggesting local retroviral spreading and/or clonal origin. Subsequent demonstration of proviral integration was confirmed by the
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