The inadequacies of each viral vector system are illustrated in Table II. The negative attributes of one vector, however, generally emphasize the positive attributes of another. Thus most of the criteria defined for a hypothetical perfect gene therapy might actually be met by considering defined properties of the currently available vectors defined in Table II. Hence, although at present no individual virus system alone can meet all the criteria, current research is focusing on combining individual viral properties into single vector constructs, termed "hybrid" or "chimeric" vectors.
Adenoviral vectors are currently the major vector choice for a variety of clinical disorders, despite the limited efficacy due to the transient nature of the vector. Mechanisms of enhancing the pharmaceutical properties of Ad vectors are thus highly desirable. The incorporation of other viral vector functions that could enhance the duration of Ad-directed transgene expression and/or target the vectors to a specific disease tissue would be extremely beneficial. In essence, whether the aim is to kill or cure the target cell, a vector encompassing the advantageous properties of high titer, broad host range, and infectivity of an Ad vector, together with the low immunogenicity and potential for long-term stable expression of a retrovirus, AAV, or EBV vector would be extremely useful for gene therapy for a wide range of genetic and acquired disorders. Hence the main focus of this chapter is to review the properties of other viral vectors which have been utilized to generate hybrid adenoviral vectors in the aim of enhancing vector efficacy in the clinic.
A. Are Hybrid Vectors Truly New Technology?
The formation of hybrid adenoviruses is not a new technology and has been extensively reported to occur naturally in nature. Adenoviral/simian virus 40 (SV40) hybrids have been documented to occur in nature [43, 44]. Although human adenoviruses do not normally replicate in primate cells, upon coinfection with SV40, Ad genomes acquired sequences from the SV40 genomes (large T antigen) which permitted replication and assembly of hybrid genomes into wild-type Ad capsid particles . Additionally it may be that the helper-dependent AAV genome represents a segment of an extinct or undiscovered virus that was selected upon coinfection with an Ad or an HSV. Perhaps the parental virus was too virulent to coexist in a human host, thereby explaining the nonpathogenic nature of the dependovirus.
The development of hybrid viral vectors is fundamentally not a new technology in gene therapy. Since the dawn of gene therapy, scientists have utilized alternative ds-acting sequences from other viruses, specifically promoters and enhancers, to drive transgene expression. Most significantly, the cytomegalovirus (CMV) immediate-early promoter and enhancer has been utilized in almost every viral vector reported to date and is well characterized as an extremely strong constitutive promoter in most tissues [45, 46]. Other well utilized viral promoters have included the Rous sarcoma virus (RSV) LTR promoter, the SV40 early promoter, hepatitis B virus (HBV), and the EBV promoter [45, 46]. Additionally, application of the picornaviral functions of "cap-independent" initiation of translation has also been extensively exploited in viral vectors. These translational regulatory elements, termed internal ribosomal entry site (IRES) sequences, enable bicistronic expression from a single mRNA transcript . The application of these elements greatly complemented the limited insert capacities of viral vectors, thereby negating the need for separate promoters to drive two transgene cassettes.
Retroviral vectors have been studied in hybrid vector systems since the early 1980s, "pseudotyping" them with functions from other retroviral vectors. Specifically heterotropic viral glycoproteins from other retroviral env genes have been stably incorporated into MoMuLV vector particles. The incorporation of vesicular somatic virus G (VSV-G) glycoprotein , gibbon ape leukemia virus (GALV) and HIV-1 glycoproteins  into murine leukemia virus particles has been reported. These hybrid MoMuLV virions attain the tropism of the pseudotyped env proteins, retargeting or broadening the host range of the MoMuLV vector. Additionally, incorporation of VSV-G env has been demonstrated to increase the stability of the virions, enabling higher titer-yielding purification techniques to be applied [50, 51]. Hybrid retroviral vectors have also been constructed, incorporating different cis-acting elements contained in the U3 region of the LTR, which direct the transcriptional activity of the virus. Replacement of these U3 regulatory elements can impart tissue-specific transcriptional activity on the RV vector [52, 53]. Hence the concept of hybrid vectors is not a new technology, but the new strategies proposed could vastly expand the repertoire of viral vectors available to the clinic.
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