Targeting AAV vectors
Authors: Andrew H Baker
Journal: Molecular Therapy (2003)

One of the many hurdles that face gene therapists is the requirement to optimize gene delivery systems in such a way that both efficient and selective transgene production is achieved in vivo within a chosen cell or tissue. Exploiting native vector tropism can sometimes achieve this goal irrespective of additional modifications. For example, adenoviral infection of liver hepatocytes following systemic administration or adeno-associated viral (AAV) infection of skeletal muscle intramuscularly allows high-level, site-selective transgene production. However, there remains a plethora of diseases in which the target cell population is poorly transduced in vivo by available vector systems, raising critical issues over feasibility and safety for the proposed genetic medicine. Classical examples include cancer and a variety of cardiovascular disorders. In this issue of Molecular Therapy Shi and Bartlett [1Shi and Bartlett (2003). Mol. TherGoogle Scholar] engineer a novel AAV serotype 2 (AAV-2) vector that mediates enhanced transduction of cancer cells that are normally refractory to AAV-2 transduction. Vector targeting has become a major focus in many areas of gene therapy. Targeting can be achieved at the level of the vector:cell interaction (transductional targeting) and at the level of transgene regulation (transcriptional targeting). Naturally, tethering of the vector to the cell surface is the principle mechanism dictating vector tropism and as such is the initial focus of modification to reengineer new tropism, either to increase efficiency of gene transfer or to introduce selectivity or both, depending on the downstream application. As the route of vector administration and the list of target pathologies are highly diverse, transductional targeting remains a major technical challenge and, in the case of viral vectors, requires detailed knowledge of capsid structure and function as well as availability of suitable targeting agents. Traditionally, retroviral and adenoviral vectors have been at the forefront of developments in vector targeting, with a variety of techniques being documented, including vector pseudotyping, use of bispecific antibodies, and insertion of targeting peptides into viral capsids. The strategy may also require the blockade of native tropism and/or simultaneous use of transcriptional targeting. AAV-2 vectors possess a highly favorable safety profile and have demonstrated unique potential in certain models of disease, but their applicability is limited due to the restricted range of cells that they efficiently transduce. Although use of alternate AAV serotypes can enhance gene delivery to certain tissues, this does not generate defined tropism selective for the target disease. Retargeting AAV-2 vectors may allow this. Native AAV-2 tropism is depicted in Fig. 1A. To date, transductional retargeting of AAV-2 has been achieved in vitro using antibodies [2Bartlett J.S. Kleinschmidt J. Boucher R.C. Samulski R.J. Targeted adeno-associated virus vector transduction of non-permissive cells mediated by a bi-specific F(ab′γ) (2) antibody.Nat. Biotechnol. 1999; 17: 181-186Crossref PubMed Scopus (198) Google Scholar, 3Yang Q. et al.Development of novel cell surface CD34-targeted recombinant adeno associated virus vectors for gene therapy.Hum. Gene Ther. 1998; 9: 1929-1937Crossref PubMed Scopus (100) Google Scholar] and peptides [4Shi W. Arnold G.S. Bartlett J.S. Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors.Hum. Gene Ther. 2001; 12: 1697-1711Crossref PubMed Scopus (134) Google Scholar, 5Wu P. et al.Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.J. Virol. 2000; 74: 8635-8647Crossref PubMed Scopus (321) Google Scholar, 6Grifman M. et al.Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids.Mol. Ther. 2001; 3: 964-975Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7Girod A. et al.Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.Nat. Med. 1999; 9: 1052-1056Google Scholar, 8Nicklin S.A. et al.Efficient and selective AAV-2 mediated gene transfer directed to human vascular endothelial cells.Mol. Ther. 2001; 4 (doi:10.1006/mthe.2001.0424): 174-181Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar], the latter being more successful. Appropriate sites for peptide insertion in the AAV-2 capsid have been mapped, some of which accommodate peptide insertion without detriment to virion assembly. Targeting with peptides has three major advantages over other approaches. First, utilization of peptide phage display technology allows the identification of highly efficient and selective targeting peptides for diverse cell types (without the necessity for prior knowledge of the target receptor). Second, targeting peptides are small and can be genetically inserted into viral capsids to produce single-component vector systems. Third, some evidence suggests that native AAV tropism can be reduced by targeting-peptide insertion, presumably through steric hindrance imposed by the inserted peptide on the heparan sulfate proteoglycan (HSPG)-binding domain [8Nicklin S.A. et al.Efficient and selective AAV-2 mediated gene transfer directed to human vascular endothelial cells.Mol. Ther. 2001; 4 (doi:10.1006/mthe.2001.0424): 174-181Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar]. In practice, retargeting to alternate receptors may or may not remove the requirement for the virion to internalize using its native coreceptors and is likely to be entirely dependent on the nature of the targeted receptor (Fig. 1B). Shi and Bartlett [1Shi and Bartlett (2003). Mol. TherGoogle Scholar] utilize these concepts and document the ability to retarget AAV-2 using the RGD4C peptide in vitro and apply the targeting principle using an in vivo tumor model. The RGD4C peptide, isolated by Pasqualini and co-workers [9Pasqualini R.E. Koivvunen E. Ruoslahti E. Alpha v integrins as receptors for tumor targeting by circulating ligands.Nat. Biotechnol. 1997; 15: 542-546Crossref PubMed Scopus (702) Google Scholar], has proven highly efficient for adenoviral retargeting by broadening tropism and thereby enhancing transduction of many cell types [10Dmitriev I. et al.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]. Based on this proven capability, the authors use RGD4C to optimize peptide insertion sites and associated linker/scaffolds for compatibility with virion assembly, maintenance of titer, and retargeting capacity. Encouragingly, the optimal site of insertion was at position 588 in the surface-exposed GH loop of VP3, consistent with the locale within the virion used by other groups and supported by the recently solved crystal structure of AAV-2 [11Xie Q. et al.The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.Proc. Natl. Acad. Sci. USA. 2002; 99: 10405-10410Crossref PubMed Scopus (460) Google Scholar]. From in vitro studies, the authors demonstrate that infection of target cells is mediated directly through the inserted RGD4C peptide interacting with target integrins in a manner independent of HSPG binding. In vivo potential was defined by enhancement of transduction in a murine model of ovarian cancer following ip administration and is the first such demonstration of in vivo AAV retargeting. This study, together with previous reports, provides ample evidence to suggest that reengineering of AAV-2 vectors is a feasible, efficient, and applicable concept. To envisage the utilization of this technology in a broader sense will require the availability of a range of suitable homing peptides. These need to be compatible with AAV-2 capsid structure and maintain their unique targeting fidelity when exposed at the virion surface. The availability of such a range of peptides is limited. While the RGD4C peptide may be useful for local gene delivery, its ability to target multiple cell types may limit its application in vivo, especially for cytotoxic strategies in which localized or even systemic vector dissemination may occur. Like other vector systems it will be important, therefore, to develop the targeting concept to generate AAV-2 vectors that are systemically injectable, devoid of native tropism, and with the capacity to “home-in” on the target site in vivo. This may require additional mutations in the AAV-2 cap gene to eliminate hepatic sequestration. Since AAV-2-mediated transduction can also be negatively impacted by intracellular mechanisms such as proteasome degradation [12Duan D. Yue Y. Yan Z. Engelhardt J.F. Endosomal processing limits gene transfer to polarised airway epithelia by adeno-associated virus.J. Clin. Invest. 2000; 105: 1573-1587Crossref PubMed Scopus (306) Google Scholar] and second-strand synthesis [13Qing K. Hansen J. Weigel-Kelley K. Tan M. Zhou S. Srivastava A. Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression.Virology. 2001; 75: 8968-8976Crossref Scopus (107) Google Scholar], it will be important to define how modification of tropism at the cell surface influences routing of the virion to the nucleus. In summary, AAV vectors currently offer unique potential to treat pathologies that exploit the native tropism of the virion. Manipulation of the AAV-2 capsid by peptide insertion will broaden the range of diseases applicable to AAV-2-mediated gene therapy.