Rational Design of Viral Vectors Based on Rigorous Analysis of Capsid Structures
Authors: David T. Curiel
Journal: Molecular Therapy (2000)

The design of viral vectors has been predicated upon a central assumption—exploitation of viral transduction mechanisms to effect efficient transfer of therapeutic genes. Implicit in these initial efforts was the notion that viral tropism, such as it was understood, was an intrinsic parameter which could likewise be expropriated for delivery of therapeutic transgenes. On this basis, the principal strategy embodied in the initial generation of viral vectors involved incorporation of therapeutic transgenes within the context of the genome of the parent virus. As such, this approach engendered vector design whereby parent viral tropism dictated vector tropism. The limitations of this logic of vector design have become amply apparent in the context of the human clinical trials carried out to date. In this regard, limited vector efficiencies in these endeavors have represented a central limitation to the gene therapy field as a whole. A concise articulation of this understanding was advanced in the Orkin–Motulsky report whereby it was recommended that, "To confront the major outstanding obstacles to successful somatic gene therapy, greater focus on basic aspects of gene transfer is required. Such efforts need to be applied to improving vectors for gene delivery and directing gene transfer to specific cell types." It is thus clear that there exists a substantial practical rationale to endeavor viral tropism modifications to achieve key vectorologic goals of central relevance to the gene therapy field. These human trial-directed exigencies have not, however, represented the sole impetus to alter viral tropism for vector ends. Indeed, a considerable number of efforts have been endeavored to accomplish the goal of targeted, cell-specific gene delivery based on modifications of viral vector tropism (1Douglas J.T. Curiel D.T. Targeted adenoviral vectors for cancer gene therapy.Int. J. Oncol. 1995; It: 341-348Google Scholar, 2Reynolds P.N. Curiel D.T. Strategies to adapt adenoviral vectors for gene therapy applications: Targeting and integration.in: Friedmann T. The Development of Human Gene Therapy. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1998: 111-130Google Scholar). Initial efforts in this regard capitalized on the possibility of viral pseudotyping, especially in the context of RNA viruses. These studies clearly demonstrated the principle that precise genetic engineering could achieve fundamental alterations in the biology of viral binding and entry. Further, these alterations of viral transduction physiology could be exploited for functional gene delivery in a gene therapy context (3Arai T. Matsumoto K. Saitoh K. Ui M. Ito T. Murakami M. Kanegae Y. Saito I. Cosset F.L. Takeuchi Y. Iba H. A new system for stringent, high-titer vesicular stomatitis virus G protein-pseudotyped retrovirus vector induction by introduction of Cre recombinase into stable prepackaging cell lines.J. Virol. 1998; 72: 1115-1121PubMed Google Scholar, 4Wilson C. Reitz M.S. Okayama H. Eiden M.V. Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus.J. Virol. 1989; 63: 2374-2378PubMed Google Scholar, 5Johnson J.E. Schnell M.J. Buonocore L. Rose J.K. Specific targeting to CD4+ cells of recombinant vesicular stomatitis viruses encoding human immunodeficiency virus envelope proteins.J. Virol. 1997; 71: 5060-5068Crossref PubMed Scopus (104) Google Scholar). While these studies provided proof-of-principle that viral, and thus vector, tropism could be modified, specificity aspects, however, were still subservient to native viral tropisms. Natural diversity clearly did not provide a range of biologic flexibility compatible with the broad range of gene therapy requirements. Based on these considerations, attempts to engineer precise tropism specificities have sought to advance the conceptual gains achieved in the context of pseudotyping. Such efforts were first endeavored for retroviral vectors via directed alterations in the env glycoprotein to allow cell-specific binding, and thus transduction (6Nilson, B. H., Morling, F. J., Cosset, F. L., and Russell, S. J.Targeting of retroviral vectors through protease–substrate interactions. Gene Ther.3: 280–286.Google Scholar, 7Kabat D. Targeting retroviral vectors to specific cells.Science. 1995; 269: 417Crossref PubMed Scopus (16) Google Scholar, 8Cosset F.L. Russell S.J. Targeting retrovirus entry.Gene Ther. 1996; 3: 946-956PubMed Google Scholar). Of note, these genetic modifications were endeavored in the absence of a full and complete structural understanding of the target viral protein. As such, these attempts to alter the function of key viral components have been largely empirical. The limited success of these efforts has thus belied the notion that empiric alterations of key viral vectors could be trivially achieved; these attempts to alter key viral proteins for tropism alterations have only served to reveal the complex interplay of viral entry functions relevant to engineered vector design. Clearly, a further understanding of key viral structural elements is an absolute predicate of productive alterations of these components to achieve functional modifications of vector tropism. An alternative vector system, the adenovirus, has provided an alternate paradigm whereby key structural information has been, in some instances, available in advance of tropism modification efforts. Initially, attempts to modify tropism sought to append ligands at the carboxy terminus of the fiber capsid protein (9Michael S.I. Hong J.S. Curiel D.T. Engler J.A. Addition of a short peptide ligand to the adenovirus fiber protein.Gene Ther. 1995; 2: 660-668PubMed Google Scholar). These attempts were largely empiric; this site proved to be suboptimal for viral tropism modification based on unanticipated structural factors (10Wickham T.J. Tzeng E. Shears L.L. Roelvink P.W. Li Y. Lee G.M. Brough D.E. Lizonova A. Kovesdi I. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.J. Virol. 1997; 71: 8221-8229Crossref PubMed Google Scholar). Subsequent attempts to alter adenoviral tropism benefited from consideration of available structural data with respect to capsid proteins involved in adenoviral binding and entry (11Henry L.J. Xia D. Wilke M.E. Deisenhofer J. Gerard R.D. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.J. Virol. 1994; 68: 5239-5246PubMed Google Scholar). On this basis, several lines of data, including structural considerations, suggested the HI loop of the fiber knob as a propitious locale for incorporation of heterologous peptides. The realization of the practical basis of exploiting this locale for the incorporation of targeting motifs, by our group and others, has dramatically improved efficiency attributes of this vector (12Dmitriev I. Krasnykh V. Miller C.R. Wang M. Kashentseva E. Mikheeva G. Belousova N. Curiel D.T. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism.J. Virol. 1998; 72: 9706-9713Crossref PubMed Google Scholar, 13Vanderkwaak T.M. Wang M. Navarro J.G. Rancourt C. Dmitriev I. Krasnykh V. Barnes M. Siegal G.P. Alvarez R.D. Curiel D.T. An advanced generation of adenoviral vectors selectively enhances gene transfer for ovarian cancer gene therapy approaches.Gyn. Oncol. 1999; 74: 227-234Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 14Kasono K. Blackwell J.L. Douglas J.T. Dmitriev I. Strong T.V. Reynolds P. Kropf D.A. Carroll W.R. Peters G.E. Bucy R.P. Curiel D.T. Krasnykh V. Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.Clin. Cancer Res. 1999; 5: 2571-2579PubMed Google Scholar). Such engineered enhancement in vector performance has likewise been configured into the design of human clinical trials seeking to circumvent the vector efficiency limits noted in previous human protocols. Thus, in this instance, the lesson has been clear—structural data, even of a limited nature, have allowed dramatic advancements in the ability to achieve engineered structure/function changes of vectorologic utility. Likewise, available structural data have allowed additional functional gain in the adenoviral vector context. In this regard, attempts to ablate native adenoviral tropism have advanced on this basis. Of note, capsid modification has relevance over-and-above tropism alterations. Specifically, directed alterations in immunodominant epitopes have allowed the derivation of adenoviral vectors which possess a relative ability to avoid inactivation by preexistent humoral immunity. In addition, capsid structure clearly has key relevance to viral biodistribution, clearance by the reticuloendothelial system, and interaction with specific and nonspecific humoral factors. As such, fundamental aspects of host–vector interaction may be ultimately understood in terms of capsid structure/function analysis. It is thus clear that engineered alterations in the vial capsid will accrue a variety of direct benefits from the vectorology standpoint. These recognitions have predicated the need for rigorous and complete characterizations of key viral structures relevant to the axis of host–vector interaction. The current issue of Molecular Therapy contains such a study in the article by Rux et al. (15Ruex J.J. Burnett R.M. Type-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexon.Mol. Ther. 1999; 1: 000Google Scholar). This work presents an advanced characterization of the hexon capsid protein. As the authors note, the enhanced definition they provide of hexon structure will allow a more rational approach to directed alternations of this capsid protein toward vector-directed endpoints. Such ends may include tropism alterations, immune evasion strategies, and other, as yet unrealized gains, accruing to rational modifications of viral structure. The lessons inherent herein are clear—"structure precedes function" as relates to modifying viral proteins for vectorologic gain. Indeed, when such vector design efforts can be carried out with such a priori knowledge, it is truly as for the man having turned from darkness into the light. The gains, under such circumstances, can indeed be dazzling.