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Retargeting gene delivery using surface-engineered retroviral vector particles
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Retargeting gene delivery using surface-engineered retroviral vector particles Dimitri Lavillette*, Stephen J Russell† and François-Loïc Cosset*‡ Retroviral vectors with the capacity to deliver transgenes to specific tissues are expected to be of great value for various gene transfer applications in vivo. Initial attempts to modify vector host-range by the insertion of ligands on their surface glycoproteins have frequently failed, essentially owing to the impairment of the fusogenicity of the vector particles bound to the targeted cell-surface molecules. Several strategies aimed to recover the fusogenic activity of surface-engineered vector particles have recently been explored and have given rise to novel concepts in the field. Addresses *Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, Unité de Virologie Humaine, INSERM U412, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, 69364 Lyon Cedex 07, France † Molecular Medicine Program, Guggenheim 18, Mayo Clinic, 200 First Street, SW Rochester, MN 55905, USA; e-mail: [email protected] ‡ e-mail: [email protected] Current Opinion in Biotechnology 2001, 12:461–466 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ALV avian leukosis virus EGF epidermal growth factor HA hemagglutinin MLV murine leukemia virus MMP matrix metalloprotease SNV spleen necrosis virus
Introduction Retroviral vectors are powerful gene delivery vectors that have been derived from many types of retroviruses. Their nucleocapsid, which contains the genetic material, is wrapped by a cell-derived bilipidic membrane in which the viral glycoproteins are inserted (Figure 1). These glycoproteins fulfil a number of critical functions such as binding of the virions to cell-surface receptors and triggering the subsequent fusion between the viral and cell membranes, a critical event that leads to the penetration of the viral nucleocapsid into the cytosol of the infected cell. The viral glycoprotein is therefore the primary focus of retroviral targeting studies which seek to achieve, through the alteration of its structure, the modification of tropism and the capacity to target specific cell types by allowing interaction with specific cell-surface receptors. Over the past few years there have been many different strategies developed to alter the host-range of retroviral envelope glycoproteins; most strategies have involved engineering the murine leukemia virus (MLV) glycoprotein (reviewed in [1,2,3•]). However, the concept of host-range modification has been, and still is, a major challenge. Indeed, although the engineering of virion binding has been relatively easy to achieve, it has been readily apparent that binding-retargeted
retroviruses were usually defective for their fusion activity. Closer examination of the fusion activity of such engineered retroviruses has revealed that fusion was actually impaired at the stage of activation of membrane fusion [4–6]. Much effort has therefore focused on strategies aimed to enforce the fusion activation of retargeted retroviruses. With this aim, several distinct methods have been developed: two-step targeting strategies which exploit an ultimate interaction with a ubiquitous retroviral receptor following specific binding; pre-activation of vector particles with bifunctional bridge proteins containing both targeting and fusion-activation moieties; and ligand display on virally incorporated pH-dependent viral glycoproteins for which fusion activation is achieved through non-specific signals. Some of the most recent advances are discussed in this review.
Protein engineering of the retroviral glycoprotein: direct targeting Retroviruses recognise a relatively limited number of cellsurface proteins as entry receptors [7]. Several retroviral glycoproteins (e.g. those of ecotropic MLV strains) do not recognise a receptor on human cells. Therefore, initial attempts to retarget retroviral tropism have consisted of the insertion of various ligand types (i.e. growth factors, hormones, peptides or single-chain antibodies) into various locations on the viral surface glycoproteins. In this way it was hoped to extend their host-range by allowing them to bind human cell-surface molecules different from the parent virus receptor (i.e. direct targeting) [3•]. Examples of insertion sites in the MLV glycoprotein that have been characterised and found to be functional, at least for retargeted binding, are shown in Figure 1. Briefly, they include modifications of the glycoprotein such as domain replacement [8], peptide insertion in prefolded domains [9,10,11•], and the display of polypeptides as additional folded domains [5,12•,13,14]. Many of these chimeric glycoproteins fold correctly, are stably incorporated on virions and allow efficient retargeted virion binding to the expected cell-surface molecules. However, upon binding to the target receptors, most of these chimeras — if not all of them — are unable to induce membrane fusion and subsequent penetration of the viral core into the cytosol. In most cases, this failure is not due to inactivation of the fusion machinery of the chimeric glycoproteins themselves, as several of these chimeras are fully functional for achieving membrane fusion once they are allowed to bind cells expressing the natural retroviral receptors [3•]. Instead, the poor fusion activity of chimeric glycoproteins is attributed to the loss of coupling between retargeted binding and fusion activation [3•,5,6]. Closer examination of the molecular mechanism that couples the binding of the natural retroviral receptors to fusion activation revealed the very
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Figure 1
TM
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Schematic representation of chimeric retroviral envelope glycoproteins. (a) Domain organisation of the MLV envelope glycoprotein precursor. SP, signal peptide. The surface subunit (SU, orange) consists of the receptorbinding domain (RBD), the proline-rich region (PRR) and the C-terminal domain (C). The transmembrane subunit (TM, multicolored) consists of an ectodomain comprising the fusion peptide (FP, red), a heptad repeat region (HR, blue) and a downstream α helix (green) followed by a transmembrane domain (tmd), a cytoplasmic tail (cyt, blue) and the R peptide. The positions of some envelope glycoprotein subdomains are shown: variable regions A (VRA) and B (VRB). The black arrows mark the positions of cleavage of the envelope precursor; the red arrows show the positions of the MLV glycoproteins that accommodate insertion/substitutions of peptides and/or polypeptides. Examples of retargeted glycoproteins, the host-range of which has been modified using (b) bifunctional protein adaptors, (c) domain substitution (e.g. RBD), (d) the insertion of small-ligand peptides and (e) the addition of large polypeptide ligands (see text for details).
(e) Interdomain spacer
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sophisticated solution adopted by retroviruses to coordinate these two functions. This coupling involves complex inter-relations between the different subdomains of the glycoprotein complex [15•,16–18]. Several other results have shown that in no way could this coupling be reconstituted via the interaction of a nonviral target receptor to a nonviral ligand, even when inserted into a fusion-competent retroviral glycoprotein [3•].
Escorting viral entry to cells co-expressing target and viral receptors Several strategies have been explored to overcome the loss of binding-to-fusion coupling imposed by the stringent fusion properties of retroviral glycoproteins. In contrast to previous methods that aimed to expand viral tropism, these
strategies are based on the display of polypeptides on retroviral glycoproteins that naturally recognise a receptor on the target cells (e.g. amphotropic MLV glycoprotein). Thus, after a phase of interaction with a specific target cell surface molecule, cellular entry of retroviral vectors carrying such chimeric glycoproteins ultimately relies on the interaction with the natural retroviral receptor, which permits efficient membrane fusion (targeting by host-range restriction). One such strategy consists of the inclusion of binding motifs on the virions [19•,20–22]. This can be achieved by the insertion of such determinants on the viral glycoprotein itself, for example, in locations that do not affect the ability to promote binding to the natural receptor and subsequent membrane fusion (Figure 1). Alternatively, the co-expression along with the wild-type glycoprotein of a second
Retargeting gene delivery using surface-engineered retroviral vector particles Lavillette, Russell and Cosset
‘escorting’ glycoprotein, which carries cell-specific binding determinants and that is usually defective for fusion, also permits the tethering of virion binding to tissues that abundantly express the target molecules [20,21]. Recent examples of vector-escorting strategies have focused on the incorporation of matrix-targeting motifs (i.e. collagen-binding peptides) on amphotropic MLV vector particles. This strategy was shown to enhance the retrovirus binding and transduction of human endothelial cells in vitro [21,23,24]. Interestingly, such vectors could localise gene delivery to sites of vascular injury in vivo in rats [23] and in the angiogenic tumor vasculature in human cancer xenografts in nude mice [25•]. Such ‘preferential’ targeting cannot be highly specific in essence and further experiments of vector biodistribution will establish whether or not the increase of affinity, achieved through the addition of binding motifs, can be sufficient to reduce significantly the leak of infectivity to non-target cells. Nevertheless, in the case of vectors derived from the avian spleen necrosis virus (SNV), a more specific targeting can be envisaged through a similar approach [19,20]. Indeed, although SNV and simian D-type viruses belong to the same receptor interference group and appear to use the same receptor molecule [7], the human allele of this receptor cannot mediate SNV entry into human cells — presumably because of its low-affinity with the SNV glycoprotein [19,20]. However, in the context of cell-surface-tethered virions that display both a wildtype glycoprotein and a chimeric glycoprotein with an engineered high-affinity binding motif, it is likely that the SNV glycoprotein could interact with the human receptor and may thus promote membrane fusion in a rather restricted manner [19,26].
Retroviral display of cell-specific blocking domains to restrict tropism Other strategies that, following a step of targeted binding, exploit an ultimate interaction with the viral receptor have been based on the discovery that some ligand–receptor pairs have the capacity to sequester cell-bound retargeted virions, thus impairing their infectivity [3•,5,27]. For example, the display of epidermal growth factor (EGF) on the amphotropic MLV glycoprotein results in a dramatic inhibition of infectivity on EGF-receptor-positive cells. Such a sequestration can be competitively abrogated by adding the displayed ligand as soluble polypeptide [5]. The blocking effect exerted by the displayed EGF occurs even if the amphotropic retroviral receptor is co-expressed on the target cells and despite the full infectivity of such retroviruses on EGF-receptor-negative cells [5,27]. Thus EGF-displaying amphotropic vectors can efficiently infect EGF-receptor-negative cells, but are not infectious on EGF-receptor-positive cells. Receptor-mediated virion sequestration has been documented mainly — though not exclusively — for molecules that belong to the family of tyrosine kinase receptors and can be applied to target gene delivery to cells that express only the viral receptor in mixed cell populations (inverse targeting) [27]. Intravenous inoculation in mice of non-targeted lentiviral vectors, carrying wild-type glycoproteins, leads to maximal
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reporter gene activity in liver and spleen with moderate or minimal expression in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, EGF-displaying vectors inoculated intravenously are expressed maximally in the spleen with very low-level expression detectable in EGFreceptor-rich liver cells. Liver transduction by the EGF-displaying vector can be restored by pre-treating the animals with soluble EGF, suggesting that these vectors are inversely targeted to spleen cells [28••]. These results are of primary importance for several reasons: they demonstrate that it is possible to generate lentiviral vectors carrying retargeted glycoproteins; they address the biodistribution of vectors after systemic inoculation; and they establish the feasibility of retargeting gene delivery in vivo. On a parallel path, capitalising on the sequestration properties of some cell-surface molecules expressed on target cells, a specific gene delivery can also be envisaged [29–32]. This depends on the definition of a molecular device that allows the virions to escape the sequestering receptor. This can be achieved by inserting, between the displayed virionsequestrating ligand and the viral glycoprotein, peptide substrates that are cleaved by cell-surface-specific proteases (Figure 1). For example, EGF-displaying amphotropic retroviruses carrying a matrix metalloprotease (MMP) cleavage site could preferentially infect EGF-receptorpositive MMP-rich target cells in vitro [30] and could discriminate between MMP-rich and MMP-poor tumour xenografts implanted into nude mice [33]. Masking of the retroviral envelope functions can also be achieved in a very different and more general manner by using trimerising cleavable protein domains. Such polypeptides can block infection in a way that is independent of the variable inclination of some displayed ligands to induce receptor-mediated virus sequestration. For example, the N-terminal expression of the trimerising extracellular domain of CD40 ligand (amino acids 116–261) on the amphotropic MLV glycoprotein, by inducing a constraint on the glycoprotein structure, hinders receptor binding and/or fusion activation and prevents infection [34]. Relief of the block in infection can be achieved by inserting, between such virus entry blocking domains and the viral glycoprotein, a protease cleavage site for which a specific cell-surface protease is expressed on target cells [34]. These results have therefore opened the possibility that proteases on the cell surface, rather than receptors, could be used to target gene delivery, owing to the specific expression of many proteases at, or close to, the surface of tumor cells. Retroviral vectors that can be activated by cell-surface-associated host proteases involved in carcinogenesis can be generated and may also be used to identify novel proteases, by using random peptide display libraries inserted between the infection-blocking domain and the viral glycoprotein [35].
Bifunctional fusogenic adapter molecules Studies aimed at crosslinking vector particles and target cellsurface molecules by using, for example, ligand-conjugated
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antibodies against the viral glycoprotein were among the first approaches explored to establish the proof-of-concept of retargeted gene delivery [36]. Despite the relative inefficiency of these early results [37], interest in these strategies has recently been revived with the development of more sophisticated adapter molecules that can simultaneously retarget virion binding and trigger conformational changes in the glycoprotein of retroviruses that belong to the group of avian leukosis viruses (ALVs) [38]. Such ‘intelligent’ protein adapters consist of recombinant polypeptides in which ligands are fused to soluble forms of the receptors for ALVs, which are single-spaning transmembrane molecules [7]. Thus, these bifunctional bridge proteins bind virions to specific cell-surface molecules and the retroviral receptor moiety activates viral entry into target cells. Proof-of-principle for this elegant targeting strategy, which does not require molecular engineering of the viral glycoprotein, has been established in vitro for adapter molecules that carry EGFreceptor-binding determinants, such as EGF itself or an anti-EGF receptor single-chain antibody [39]. The bridge proteins could be either preloaded on target cells before gene transfer or, interestingly, preloaded on ALV vector particles [40•]. Thus, the rather restricted host-range of these avian retroviral vectors could be expanded to mammalian cells.
Ligand display on non-retroviral glycoproteins For several reasons, it is anticipated that it will not be possible to develop bifunctional fusogenic adapter molecules for vectors coated with MLV glycoproteins. First, unlike the simple structure of ALV receptors, the complex topology of the MLV receptor molecules [7] precludes their functional association with ligands as recombinant soluble polypeptides. Second, the fusion activation of MLV retroviral glycoproteins induced by receptor-binding seems to be connected with essential downstream cellular pathways [41,42]. It seems difficult therefore to overcome the very specific fusion activation mechanism of MLV glycoproteins in direct retargeting strategies. In contrast, the ability of the glycoproteins of some membrane-enveloped viruses to fuse in the low-pH endosomes associated with the high turnover rate of most cell-surface molecules provides an alternative basis for targeting strategies. Examples of glycoproteins that use a pH-dependent fusion activation pathway and that are efficiently assembled on retroviral particles involved those derived from vesicular stomatitis virus, semliki forest virus, lymphocytic choriomeningitis virus, sindbis virus and influenza viruses. The fusion mechanism of influenza virus hemagglutinin (HA) glycoprotein has been extensively studied and does not appear to depend primarily on binding to the HA receptor [43]. In fact, the activation of the fusion functions of this viral glycoprotein is achieved through acidification in the endosome; the virions are trafficked to the endosome following binding to the influenza virus receptor, sialic acid, a ubiquitous sugar moiety linked to most cell-surface glycoproteins. Thus the physiological signals that trigger the membrane-fusion properties of HA are much less specific than those of retroviral envelope glycoproteins and can activate HA proteins independently of their binding to
receptors. Co-expression of fusion-defective MLV-based retargeted envelope glycoproteins with wild-type HA or with HA molecules that bear mutations that prevent sialic acid binding have been shown to stimulate gene delivery in target cells, albeit at low efficiency [44,45]. Several ligand types have also been functionally displayed on HA proteins, either as N-terminal extensions of HA or by insertion into the HA structure itself [46•,47]. Recombinant HA glycoproteins that displayed N-terminal ligands were correctly expressed and processed, and efficiently incorporated into MLV retroviral particles. This observation indicated that the N-terminal insertion of large polypeptides did not alter the conformation of HA chimeras [46•]. As expected from the pH-dependent fusion properties of HA, virions generated with the HA chimeric glycoproteins had wild-type fusion activity when used to infect cells expressing the target surface molecules. Moreover, the host-range of MLV vector particles coated with chimeric HA proteins could be selectively re-addressed to cells expressing the expected target cell surface molecules [46•]. As HA proteins can efficiently pseudotype primate lentiviral vectors [48, 49], these results open the way to numerous in vivo applications in gene transfer areas. Functional display of polypeptides on lentivirus vectors has also been recently established for an alternative pH-dependent glycoprotein [50]. In this study, an IgG-binding domain of protein A (domain ZZ), derived from Staphylococcus aureus, was inserted in the E2 surface glycoprotein of sindbis virus. In a manner similar to the display of the ZZ domain on HA glycoproteins [46•], gene delivery by retroviral vectors carrying the E2 chimeric glycoprotein could be retargeted in the presence of target cell surface specific antibodies [50].
Conclusions The initial expectations raised by the early discovery that it was possible to specify the binding of retroviral vectors to particular cell types was severely challenged as it soon appeared that several basic problems needed to be overcome before efficient retargeted retroviral vectors, suitable for in vivo gene delivery, could become available. Recent results have helped to elucidate the basis of the failure to modify the tropism of genetically engineered retroviral glycoproteins in host-range extension strategies (i.e. poor fusion activation induced by retargeted binding). In the meantime, several innovative approaches have been explored to overcome this problem and have given rise to novel concepts in the field. Several very different solutions have been found to improve recruitment of the fusion properties of retargeted retroviral vectors and have provided promising results in preliminary evaluations in vivo. The concept of retargeted retroviral vectors was recently questioned by the finding that, at least in tissue culture systems, (retargeted) retroviruses can non-specifically adsorb on adherent cell types, even in the absence of a (target) receptor for their (chimeric) glycoproteins [51,52]. Although not yet supported by parallel in vivo data, these studies raise the question of the potential utility of modifying the viral
Retargeting gene delivery using surface-engineered retroviral vector particles Lavillette, Russell and Cosset
tropism. However, although non-specific adhesion may possibly lead to wastage of vector particles on non-target cells after inoculation in vivo, it is clear that it does not result in productive infection. Moreover, one of the best examples of a cell-type-specific virus, HIV-1, is known to adhere to a variety of cell-surface molecules on different non-target cells [53]. Nevertheless, this does not prevent specific interaction and infection with the specific target cells.
Acknowledgements Work in the laboratory of the authors is supported by Agence Nationale pour la Recherche contre le SIDA (ANRS), the European Community, Association Franco-Israélienne pour la Recherche Scientifique et Technologique (AFIRST), Association Française contre les Myopathies (AFM), Association pour la Recherche contre le Cancer (ARC), Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé Et de la Recherche Médicale (INSERM). This review is dedicated to the memory of Pierrick Thoraval (1960–2000), friend and colleague.
13. Lorimer IA, Lavictoire SJ: Targeting retrovirus to cancer cells expressing a mutant EGF receptor by insertion of a single chain antibody variable domain in the envelope glycoprotein receptor binding lobe. J Immunol Methods 2000, 237:147-157. 14. Russell SJ, Hawkins RE, Winter G: Retroviral vectors displaying functional antibody fragments. Nucleic Acids Res 1993, 21:1081-1085. 15. Barnett AL, Davey RA, Cunningham JM: Modular organization of the • Friend murine leukemia virus envelope protein underlies the mechanism of infection. Proc Natl Acad Sci USA 2001, 98:4113-4118. This article, which addresses the molecular coupling between receptor binding and fusion activation in the MLV glycoprotein, may elucidate the mechanism of one of the first described retargeted retroviral glycoproteins which was targeted to the erythropoietin receptor (see [8]). 16. Lavillette D, Maurice M, Roche C, Russell SJ, Sitbon M, Cosset F-L: A proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes. J Virol 1998, 72:9955-9965. 17.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Buchholz CJ, Stitz J, Cichutek K: Retroviral cell targeting vectors. Curr Opin Mol Ther 1999, 1:613-621.
2.
Karanavas G, Marin M, Salmons B, Gunzburg WH, Piechaczyk M: Cell targeting by murine retroviral vectors. Crit Rev Oncol Hematol 1998, 28:7-30.
3. Russell SJ, Cosset F-L: Modifying the host range properties of • retroviral vectors. J Gene Med 1999, 1:300-311. A recent exhaustive review on cell targeting by retroviruses. 4.
Benedict CA, Tun RY, Rubinstein DB, Guillaume T, Cannon PM, Anderson WF: Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus–cell fusion. Hum Gene Ther 1999, 10:545-557.
5.
Cosset F-L, Morling FJ, Takeuchi Y, Weiss RA, Collins MKL, Russell SJ: Retroviral retargeting by envelopes expressing an N-terminal binding domain. J Virol 1995, 69:6314-6322.
6.
Zhao Y, Zhu L, Lee S, Li L, Chang E, Soong NW, Douer D, Anderson WF: Identification of the block in targeted retroviralmediated gene transfer. Proc Natl Acad Sci USA 1999, 96:4005-4010.
7.
Sommerfelt MA: Retrovirus receptors. J Gen Virol 1999, 80:3049-3064.
8.
Kasahara N, Dozy AM, Kan YW: Tissue-specific targeting of retroviral vectors through ligand–receptor interactions. Science 1994, 266:1373-1376.
9.
Battini JL, Danos O, Heard JM: Definition of a 14-amino-acid peptide essential for the interaction between the murine leukemia virus amphotropic envelope glycoprotein and its receptor. J Virol 1998, 72:428-435.
10. Valsesia-Wittmann S, Drynda A, Deleage G, Aumailley M, Heard J-M, Danos O, Verdier G, Cosset F-L: Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors. J Virol 1994, 68:4609-4619.
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Lavillette D, Boson B, Russell S, Cosset F-L: Membrane fusion by murine leukemia viruses is activated in cis or in trans by interactions of the receptor-binding domain with a conserved disulfide loop at the carboxy-terminus of the surface glycoproteins. J Virol 2001, 75:3685-3695.
18. Zhao Y, Lee S, Anderson WF: Functional interactions between monomers of the retroviral envelope protein complex. J Virol 1997, 71:6967-6972. 19. Engelstadter M, Bobkova M, Baier M, Stitz J, Holtkamp N, Chu TH, • Kurth R, Dornburg R, Buchholz CJ, Cichutek K: Targeting human T cells by retroviral vectors displaying antibody domains selected from a phage display library. Hum Gene Ther 2000, 11:293-303. This article describes the selection of antibody fragments from a phage-display library that are able to mediate efficient and potential gene transfer into human T cells. 20. Jiang A, Chu TH, Nocken F, Cichutek K, Dornburg R: Cell-typespecific gene transfer into human cells with retroviral vectors that display single-chain antibodies. J Virol 1998, 72:10148-10156. 21. Hall FL, Gordon EM, Wu L, Zhu NL, Skotzko MJ, Starnes VA, Anderson WF: Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein. Hum Gene Ther 1997, 8:2183-2192. 22. Martin F, Kupsch J, Takeuchi Y, Russell S, Cosset F-L, Collins M: Retroviral vector targeting to melanoma cells by single-chain antibody incorporation in the envelope. Hum Gene Ther 1998, 9:737-746. 23. Hall FL, Liu L, Zhu NL, Stapfer M, Anderson WF, Beart RW, Gordon EM: Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor. Hum Gene Ther 2000, 11:983-993. 24. Liu L, Anderson WF, Beart RW, Gordon EM, Hall FL: Incorporation of tumor vasculature targeting motifs into Moloney murine leukemia virus env escort proteins enhances retrovirus binding and transduction of human endothelial cells. J Virol 2000, 74:5320-5328. 25. Gordon EM, Chen ZH, Liu L, Whitley M, Liu L, Wei D, Groshen S, • Hinton DR, Anderson WF, Beart RW et al.: Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice. Hum Gene Ther 2001, 12:193-204. This study demonstrates that a matrix-targeted retroviral vector inoculated by peripheral vein injection can enhance the delivery of a therapeutic gene in human cancer xenografts implanted in nude mice. 26. Jiang A, Dornburg R: In vivo cell-type-specific gene delivery with retroviral vectors that display single chain antibodies. Gene Ther 1999, 6:1982-1987.
•11. Wu BW, Lu J, Gallaher TK, Anderson WF, Cannon PM: Identification of regions in the Moloney murine leukemia virus SU protein that tolerate the insertion of an integrin-binding peptide. Virology 2000, 269:7-17. A comprehensive study of the properties of retroviruses generated with MLV glycoproteins bearing an insertion of heterologous peptides in various sites of the receptor-binding domain.
27.
12. Kayman SC, Park H, Saxon M, Pinter A: The hypervariable domain • of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system. J Virol 1999, 73:1802-1808. This article demonstrates that peptides and large polypeptides can be displayed at various sites of the proline-rich region of the MLV glycoprotein, without impairing its binding and fusion functions.
28. Peng KW, Pham L, Ye H, Zufferey R, Trono D, Cosset F-L, Russell SJ: •• Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors. Gene Ther 2001, in press. This study addresses the biodistribution of vectors after intravenous inoculation in mice. The results demonstrate that lentiviral vectors carrying tropism-modified glycoproteins can be preferentially retargeted to specific tissues in vivo.
Fielding AK, Maurice M, Morling FJ, Cosset F-L, Russell SJ: Inverse targeting of retroviral vectors: selective gene transfer in a mixed population of hematopoietic and nonhematopoietic cells. Blood 1998, 91:1802-1809.
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Expression vectors and delivery systems
29. Martin F, Neil S, Kupsch J, Maurice M, Cosset F-L, Collins M: Retrovirus targeting by tropism restriction to melanoma cells. J Virol 1999, 73:6923-6929.
42. Rodrigues P, Heard JM: Modulation of phosphate uptake and amphotropic murine leukemia virus entry by posttranslational modifications of PIT-2. J Virol 1999, 73:3789-3799.
30. Peng KW, FJ Morling, Cosset F-L, Murphy G, Russell SJ: A gene delivery system activatable by disease-associated matrix metalloproteinases. Hum Gene Ther 1997, 8:729-738.
43. Skehel JJ, Wiley DC: Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 2000, 69:531-569.
31. Peng KW, Morling FJ, Cosset F-L, Russell SJ: A retroviral gene delivery system activatable by plasmin. Tumor Targeting 1998, 3:112-120.
44. Hatziioannou T, Valsesia-Wittmann S, Russell S, Cosset F-L: Incorporation of fowl plague virus hemagglutinin into murine leukemia virus particles and analysis of the infectivity of the pseudotyped retroviruses. J Virol 1998, 72:5313-5317.
32. Chadwick MP, Morling FJ, Cosset F-L, Russell SJ: Modification of retroviral tropism by display of IGF-I. J Mol Biol 1999, 285:485-494. 33. Peng K-W, Vile RG, Cosset F-L, Russell SJ: Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors. Gene Ther 1999, 6:1552-1557. 34. Morling FJ, Peng K-W, Cosset F-L, Russell SJ: Masking of retroviral envelope functions by oligomerizing peptide adaptors. Virology 1997, 234:51-61. 35. Buchholz CJ, Peng K-W, Morling FJ, Zhang J, Cosset F-L, Russell SJ: In vivo selection of protease cleavage sites from retrovirus display libraries. Nat Biotechnol 1998, 16:951-954. 36. Roux P, Jeanteur P, Piechaczyk M: A versatile approach to the targeting of specific cell types by retroviruses. Proc Natl Acad Sci 1989, 86:9079-9083. 37.
Etienne-Julan M, Roux P, Carillo S, Jeanteur P, Piechaczyk M: The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker. J Gen Virol 1992, 73:3251-3255.
38. Snitkovsky S, Young JA: Cell-specific viral targeting mediated by a soluble retroviral receptor–ligand fusion protein. Proc Natl Acad Sci USA 1998, 95:7063-7068. 39. Snitkovsky S, Niederman TM, Carter BS, Mulligan RC, Young JA: A TVA-single-chain antibody fusion protein mediates specific targeting of a subgroup A avian leukosis virus vector to cells expressing a tumor-specific form of epidermal growth factor receptor. J Virol 2000, 74:9540-9545. 40. Boerger AL, Snitkovsky S, Young JA: Retroviral vectors preloaded • with a viral receptor–ligand bridge protein are targeted to specific cell types. Proc Natl Acad Sci USA 1999, 96:9867-9872. This study demonstrates that it is possible to retarget avian vectors to specific cell types through the use of a retroviral receptor–ligand fusion protein preloaded on the vector particles. 41. Kizhatil K, Albritton LM: Requirements for different components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion. J Virol 1997, 71:7145-7156.
45. Lin AH, Kasahara N, Wu W, Stripecke R, Empig CL, Anderson WF, Cannon PM: Receptor-specific targeting mediated by the coexpression of a targeted murine leukemia virus envelope protein and a binding-defective influenza hemagglutinin protein. Hum Gene Ther 2001, 12:323-332. 46. Hatziioannou T, Delahaye E, Martin F, Russell SJ, Cosset F-L: • Retroviral display of functional binding domains fused to the amino-terminus of influenza haemagglutinin. Hum Gene Ther 1999, 10:1533-1544. This article establishes that it is possible to engineer the influenza virus hemagglutinin protein by fusing ligands to its N-terminal end without affecting its fusion activity. It was then possible to use such chimeric glycoproteins to target retroviral vectors to specific cell-surface receptors. 47.
Patterson SM, Swainsbury R, Routledge EG: Antigen-specific membrane fusion mediated by the haemagglutinin protein of influenza A virus: separation of attachment and fusion functions on different molecules. Gene Ther 1999, 6:694-702.
48. Christodoulopoulos I, Cannon P: Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors. J Virol 2001, 75:4129-4138. 49. Boson B, Sandrin V, Nègre D, Cosset F-L: Efficient transduction of primary primate PBLs with SIV-derived lentiviral vectors pseudotyped with a cytoplasmic tail-modified glycoprotein derived from RD114. J Gen Med 2001, in press. 50. Morizono K, Bristol G, Xie Y-M, Kung SK-P, Chen ISC: Antibodydirected targeting of retroviral vectors via cell surface antigens. J Virol 2001, 75:8016-8020. 51. Pizzato M, Marlow SA, Blair ED, Takeuchi Y: Initial binding of murine leukemia virus particles to cells does not require specific Env–receptor interaction. J Virol 1999, in press. 52. Pizzato M, Blair ED, Fling M, Kopf J, Tomassetti A, Weiss RA, Takeuchi Y: Evidence for non-specific adsorption of targeted retrovirus vector particles to cells. Gene Ther 2001, in press. 53. Doms RW, Trono D: The plasma membrane as a combat zone in the HIV battlefield. Genes Dev 2000, 14:2677-2688.
Retargeting gene delivery using surface-engineered retroviral vector particles Dimitri Lavillette*, Stephen J Russell† and François-Loïc Cosset*‡ Retroviral vectors with the capacity to deliver transgenes to specific tissues are expected to be of great value for various gene transfer applications in vivo. Initial attempts to modify vector host-range by the insertion of ligands on their surface glycoproteins have frequently failed, essentially owing to the impairment of the fusogenicity of the vector particles bound to the targeted cell-surface molecules. Several strategies aimed to recover the fusogenic activity of surface-engineered vector particles have recently been explored and have given rise to novel concepts in the field. Addresses *Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, Unité de Virologie Humaine, INSERM U412, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, 69364 Lyon Cedex 07, France † Molecular Medicine Program, Guggenheim 18, Mayo Clinic, 200 First Street, SW Rochester, MN 55905, USA; e-mail: [email protected] ‡ e-mail: [email protected] Current Opinion in Biotechnology 2001, 12:461–466 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ALV avian leukosis virus EGF epidermal growth factor HA hemagglutinin MLV murine leukemia virus MMP matrix metalloprotease SNV spleen necrosis virus
Introduction Retroviral vectors are powerful gene delivery vectors that have been derived from many types of retroviruses. Their nucleocapsid, which contains the genetic material, is wrapped by a cell-derived bilipidic membrane in which the viral glycoproteins are inserted (Figure 1). These glycoproteins fulfil a number of critical functions such as binding of the virions to cell-surface receptors and triggering the subsequent fusion between the viral and cell membranes, a critical event that leads to the penetration of the viral nucleocapsid into the cytosol of the infected cell. The viral glycoprotein is therefore the primary focus of retroviral targeting studies which seek to achieve, through the alteration of its structure, the modification of tropism and the capacity to target specific cell types by allowing interaction with specific cell-surface receptors. Over the past few years there have been many different strategies developed to alter the host-range of retroviral envelope glycoproteins; most strategies have involved engineering the murine leukemia virus (MLV) glycoprotein (reviewed in [1,2,3•]). However, the concept of host-range modification has been, and still is, a major challenge. Indeed, although the engineering of virion binding has been relatively easy to achieve, it has been readily apparent that binding-retargeted
retroviruses were usually defective for their fusion activity. Closer examination of the fusion activity of such engineered retroviruses has revealed that fusion was actually impaired at the stage of activation of membrane fusion [4–6]. Much effort has therefore focused on strategies aimed to enforce the fusion activation of retargeted retroviruses. With this aim, several distinct methods have been developed: two-step targeting strategies which exploit an ultimate interaction with a ubiquitous retroviral receptor following specific binding; pre-activation of vector particles with bifunctional bridge proteins containing both targeting and fusion-activation moieties; and ligand display on virally incorporated pH-dependent viral glycoproteins for which fusion activation is achieved through non-specific signals. Some of the most recent advances are discussed in this review.
Protein engineering of the retroviral glycoprotein: direct targeting Retroviruses recognise a relatively limited number of cellsurface proteins as entry receptors [7]. Several retroviral glycoproteins (e.g. those of ecotropic MLV strains) do not recognise a receptor on human cells. Therefore, initial attempts to retarget retroviral tropism have consisted of the insertion of various ligand types (i.e. growth factors, hormones, peptides or single-chain antibodies) into various locations on the viral surface glycoproteins. In this way it was hoped to extend their host-range by allowing them to bind human cell-surface molecules different from the parent virus receptor (i.e. direct targeting) [3•]. Examples of insertion sites in the MLV glycoprotein that have been characterised and found to be functional, at least for retargeted binding, are shown in Figure 1. Briefly, they include modifications of the glycoprotein such as domain replacement [8], peptide insertion in prefolded domains [9,10,11•], and the display of polypeptides as additional folded domains [5,12•,13,14]. Many of these chimeric glycoproteins fold correctly, are stably incorporated on virions and allow efficient retargeted virion binding to the expected cell-surface molecules. However, upon binding to the target receptors, most of these chimeras — if not all of them — are unable to induce membrane fusion and subsequent penetration of the viral core into the cytosol. In most cases, this failure is not due to inactivation of the fusion machinery of the chimeric glycoproteins themselves, as several of these chimeras are fully functional for achieving membrane fusion once they are allowed to bind cells expressing the natural retroviral receptors [3•]. Instead, the poor fusion activity of chimeric glycoproteins is attributed to the loss of coupling between retargeted binding and fusion activation [3•,5,6]. Closer examination of the molecular mechanism that couples the binding of the natural retroviral receptors to fusion activation revealed the very
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Figure 1
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Schematic representation of chimeric retroviral envelope glycoproteins. (a) Domain organisation of the MLV envelope glycoprotein precursor. SP, signal peptide. The surface subunit (SU, orange) consists of the receptorbinding domain (RBD), the proline-rich region (PRR) and the C-terminal domain (C). The transmembrane subunit (TM, multicolored) consists of an ectodomain comprising the fusion peptide (FP, red), a heptad repeat region (HR, blue) and a downstream α helix (green) followed by a transmembrane domain (tmd), a cytoplasmic tail (cyt, blue) and the R peptide. The positions of some envelope glycoprotein subdomains are shown: variable regions A (VRA) and B (VRB). The black arrows mark the positions of cleavage of the envelope precursor; the red arrows show the positions of the MLV glycoproteins that accommodate insertion/substitutions of peptides and/or polypeptides. Examples of retargeted glycoproteins, the host-range of which has been modified using (b) bifunctional protein adaptors, (c) domain substitution (e.g. RBD), (d) the insertion of small-ligand peptides and (e) the addition of large polypeptide ligands (see text for details).
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sophisticated solution adopted by retroviruses to coordinate these two functions. This coupling involves complex inter-relations between the different subdomains of the glycoprotein complex [15•,16–18]. Several other results have shown that in no way could this coupling be reconstituted via the interaction of a nonviral target receptor to a nonviral ligand, even when inserted into a fusion-competent retroviral glycoprotein [3•].
Escorting viral entry to cells co-expressing target and viral receptors Several strategies have been explored to overcome the loss of binding-to-fusion coupling imposed by the stringent fusion properties of retroviral glycoproteins. In contrast to previous methods that aimed to expand viral tropism, these
strategies are based on the display of polypeptides on retroviral glycoproteins that naturally recognise a receptor on the target cells (e.g. amphotropic MLV glycoprotein). Thus, after a phase of interaction with a specific target cell surface molecule, cellular entry of retroviral vectors carrying such chimeric glycoproteins ultimately relies on the interaction with the natural retroviral receptor, which permits efficient membrane fusion (targeting by host-range restriction). One such strategy consists of the inclusion of binding motifs on the virions [19•,20–22]. This can be achieved by the insertion of such determinants on the viral glycoprotein itself, for example, in locations that do not affect the ability to promote binding to the natural receptor and subsequent membrane fusion (Figure 1). Alternatively, the co-expression along with the wild-type glycoprotein of a second
Retargeting gene delivery using surface-engineered retroviral vector particles Lavillette, Russell and Cosset
‘escorting’ glycoprotein, which carries cell-specific binding determinants and that is usually defective for fusion, also permits the tethering of virion binding to tissues that abundantly express the target molecules [20,21]. Recent examples of vector-escorting strategies have focused on the incorporation of matrix-targeting motifs (i.e. collagen-binding peptides) on amphotropic MLV vector particles. This strategy was shown to enhance the retrovirus binding and transduction of human endothelial cells in vitro [21,23,24]. Interestingly, such vectors could localise gene delivery to sites of vascular injury in vivo in rats [23] and in the angiogenic tumor vasculature in human cancer xenografts in nude mice [25•]. Such ‘preferential’ targeting cannot be highly specific in essence and further experiments of vector biodistribution will establish whether or not the increase of affinity, achieved through the addition of binding motifs, can be sufficient to reduce significantly the leak of infectivity to non-target cells. Nevertheless, in the case of vectors derived from the avian spleen necrosis virus (SNV), a more specific targeting can be envisaged through a similar approach [19,20]. Indeed, although SNV and simian D-type viruses belong to the same receptor interference group and appear to use the same receptor molecule [7], the human allele of this receptor cannot mediate SNV entry into human cells — presumably because of its low-affinity with the SNV glycoprotein [19,20]. However, in the context of cell-surface-tethered virions that display both a wildtype glycoprotein and a chimeric glycoprotein with an engineered high-affinity binding motif, it is likely that the SNV glycoprotein could interact with the human receptor and may thus promote membrane fusion in a rather restricted manner [19,26].
Retroviral display of cell-specific blocking domains to restrict tropism Other strategies that, following a step of targeted binding, exploit an ultimate interaction with the viral receptor have been based on the discovery that some ligand–receptor pairs have the capacity to sequester cell-bound retargeted virions, thus impairing their infectivity [3•,5,27]. For example, the display of epidermal growth factor (EGF) on the amphotropic MLV glycoprotein results in a dramatic inhibition of infectivity on EGF-receptor-positive cells. Such a sequestration can be competitively abrogated by adding the displayed ligand as soluble polypeptide [5]. The blocking effect exerted by the displayed EGF occurs even if the amphotropic retroviral receptor is co-expressed on the target cells and despite the full infectivity of such retroviruses on EGF-receptor-negative cells [5,27]. Thus EGF-displaying amphotropic vectors can efficiently infect EGF-receptor-negative cells, but are not infectious on EGF-receptor-positive cells. Receptor-mediated virion sequestration has been documented mainly — though not exclusively — for molecules that belong to the family of tyrosine kinase receptors and can be applied to target gene delivery to cells that express only the viral receptor in mixed cell populations (inverse targeting) [27]. Intravenous inoculation in mice of non-targeted lentiviral vectors, carrying wild-type glycoproteins, leads to maximal
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reporter gene activity in liver and spleen with moderate or minimal expression in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, EGF-displaying vectors inoculated intravenously are expressed maximally in the spleen with very low-level expression detectable in EGFreceptor-rich liver cells. Liver transduction by the EGF-displaying vector can be restored by pre-treating the animals with soluble EGF, suggesting that these vectors are inversely targeted to spleen cells [28••]. These results are of primary importance for several reasons: they demonstrate that it is possible to generate lentiviral vectors carrying retargeted glycoproteins; they address the biodistribution of vectors after systemic inoculation; and they establish the feasibility of retargeting gene delivery in vivo. On a parallel path, capitalising on the sequestration properties of some cell-surface molecules expressed on target cells, a specific gene delivery can also be envisaged [29–32]. This depends on the definition of a molecular device that allows the virions to escape the sequestering receptor. This can be achieved by inserting, between the displayed virionsequestrating ligand and the viral glycoprotein, peptide substrates that are cleaved by cell-surface-specific proteases (Figure 1). For example, EGF-displaying amphotropic retroviruses carrying a matrix metalloprotease (MMP) cleavage site could preferentially infect EGF-receptorpositive MMP-rich target cells in vitro [30] and could discriminate between MMP-rich and MMP-poor tumour xenografts implanted into nude mice [33]. Masking of the retroviral envelope functions can also be achieved in a very different and more general manner by using trimerising cleavable protein domains. Such polypeptides can block infection in a way that is independent of the variable inclination of some displayed ligands to induce receptor-mediated virus sequestration. For example, the N-terminal expression of the trimerising extracellular domain of CD40 ligand (amino acids 116–261) on the amphotropic MLV glycoprotein, by inducing a constraint on the glycoprotein structure, hinders receptor binding and/or fusion activation and prevents infection [34]. Relief of the block in infection can be achieved by inserting, between such virus entry blocking domains and the viral glycoprotein, a protease cleavage site for which a specific cell-surface protease is expressed on target cells [34]. These results have therefore opened the possibility that proteases on the cell surface, rather than receptors, could be used to target gene delivery, owing to the specific expression of many proteases at, or close to, the surface of tumor cells. Retroviral vectors that can be activated by cell-surface-associated host proteases involved in carcinogenesis can be generated and may also be used to identify novel proteases, by using random peptide display libraries inserted between the infection-blocking domain and the viral glycoprotein [35].
Bifunctional fusogenic adapter molecules Studies aimed at crosslinking vector particles and target cellsurface molecules by using, for example, ligand-conjugated
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antibodies against the viral glycoprotein were among the first approaches explored to establish the proof-of-concept of retargeted gene delivery [36]. Despite the relative inefficiency of these early results [37], interest in these strategies has recently been revived with the development of more sophisticated adapter molecules that can simultaneously retarget virion binding and trigger conformational changes in the glycoprotein of retroviruses that belong to the group of avian leukosis viruses (ALVs) [38]. Such ‘intelligent’ protein adapters consist of recombinant polypeptides in which ligands are fused to soluble forms of the receptors for ALVs, which are single-spaning transmembrane molecules [7]. Thus, these bifunctional bridge proteins bind virions to specific cell-surface molecules and the retroviral receptor moiety activates viral entry into target cells. Proof-of-principle for this elegant targeting strategy, which does not require molecular engineering of the viral glycoprotein, has been established in vitro for adapter molecules that carry EGFreceptor-binding determinants, such as EGF itself or an anti-EGF receptor single-chain antibody [39]. The bridge proteins could be either preloaded on target cells before gene transfer or, interestingly, preloaded on ALV vector particles [40•]. Thus, the rather restricted host-range of these avian retroviral vectors could be expanded to mammalian cells.
Ligand display on non-retroviral glycoproteins For several reasons, it is anticipated that it will not be possible to develop bifunctional fusogenic adapter molecules for vectors coated with MLV glycoproteins. First, unlike the simple structure of ALV receptors, the complex topology of the MLV receptor molecules [7] precludes their functional association with ligands as recombinant soluble polypeptides. Second, the fusion activation of MLV retroviral glycoproteins induced by receptor-binding seems to be connected with essential downstream cellular pathways [41,42]. It seems difficult therefore to overcome the very specific fusion activation mechanism of MLV glycoproteins in direct retargeting strategies. In contrast, the ability of the glycoproteins of some membrane-enveloped viruses to fuse in the low-pH endosomes associated with the high turnover rate of most cell-surface molecules provides an alternative basis for targeting strategies. Examples of glycoproteins that use a pH-dependent fusion activation pathway and that are efficiently assembled on retroviral particles involved those derived from vesicular stomatitis virus, semliki forest virus, lymphocytic choriomeningitis virus, sindbis virus and influenza viruses. The fusion mechanism of influenza virus hemagglutinin (HA) glycoprotein has been extensively studied and does not appear to depend primarily on binding to the HA receptor [43]. In fact, the activation of the fusion functions of this viral glycoprotein is achieved through acidification in the endosome; the virions are trafficked to the endosome following binding to the influenza virus receptor, sialic acid, a ubiquitous sugar moiety linked to most cell-surface glycoproteins. Thus the physiological signals that trigger the membrane-fusion properties of HA are much less specific than those of retroviral envelope glycoproteins and can activate HA proteins independently of their binding to
receptors. Co-expression of fusion-defective MLV-based retargeted envelope glycoproteins with wild-type HA or with HA molecules that bear mutations that prevent sialic acid binding have been shown to stimulate gene delivery in target cells, albeit at low efficiency [44,45]. Several ligand types have also been functionally displayed on HA proteins, either as N-terminal extensions of HA or by insertion into the HA structure itself [46•,47]. Recombinant HA glycoproteins that displayed N-terminal ligands were correctly expressed and processed, and efficiently incorporated into MLV retroviral particles. This observation indicated that the N-terminal insertion of large polypeptides did not alter the conformation of HA chimeras [46•]. As expected from the pH-dependent fusion properties of HA, virions generated with the HA chimeric glycoproteins had wild-type fusion activity when used to infect cells expressing the target surface molecules. Moreover, the host-range of MLV vector particles coated with chimeric HA proteins could be selectively re-addressed to cells expressing the expected target cell surface molecules [46•]. As HA proteins can efficiently pseudotype primate lentiviral vectors [48, 49], these results open the way to numerous in vivo applications in gene transfer areas. Functional display of polypeptides on lentivirus vectors has also been recently established for an alternative pH-dependent glycoprotein [50]. In this study, an IgG-binding domain of protein A (domain ZZ), derived from Staphylococcus aureus, was inserted in the E2 surface glycoprotein of sindbis virus. In a manner similar to the display of the ZZ domain on HA glycoproteins [46•], gene delivery by retroviral vectors carrying the E2 chimeric glycoprotein could be retargeted in the presence of target cell surface specific antibodies [50].
Conclusions The initial expectations raised by the early discovery that it was possible to specify the binding of retroviral vectors to particular cell types was severely challenged as it soon appeared that several basic problems needed to be overcome before efficient retargeted retroviral vectors, suitable for in vivo gene delivery, could become available. Recent results have helped to elucidate the basis of the failure to modify the tropism of genetically engineered retroviral glycoproteins in host-range extension strategies (i.e. poor fusion activation induced by retargeted binding). In the meantime, several innovative approaches have been explored to overcome this problem and have given rise to novel concepts in the field. Several very different solutions have been found to improve recruitment of the fusion properties of retargeted retroviral vectors and have provided promising results in preliminary evaluations in vivo. The concept of retargeted retroviral vectors was recently questioned by the finding that, at least in tissue culture systems, (retargeted) retroviruses can non-specifically adsorb on adherent cell types, even in the absence of a (target) receptor for their (chimeric) glycoproteins [51,52]. Although not yet supported by parallel in vivo data, these studies raise the question of the potential utility of modifying the viral
Retargeting gene delivery using surface-engineered retroviral vector particles Lavillette, Russell and Cosset
tropism. However, although non-specific adhesion may possibly lead to wastage of vector particles on non-target cells after inoculation in vivo, it is clear that it does not result in productive infection. Moreover, one of the best examples of a cell-type-specific virus, HIV-1, is known to adhere to a variety of cell-surface molecules on different non-target cells [53]. Nevertheless, this does not prevent specific interaction and infection with the specific target cells.
Acknowledgements Work in the laboratory of the authors is supported by Agence Nationale pour la Recherche contre le SIDA (ANRS), the European Community, Association Franco-Israélienne pour la Recherche Scientifique et Technologique (AFIRST), Association Française contre les Myopathies (AFM), Association pour la Recherche contre le Cancer (ARC), Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé Et de la Recherche Médicale (INSERM). This review is dedicated to the memory of Pierrick Thoraval (1960–2000), friend and colleague.
13. Lorimer IA, Lavictoire SJ: Targeting retrovirus to cancer cells expressing a mutant EGF receptor by insertion of a single chain antibody variable domain in the envelope glycoprotein receptor binding lobe. J Immunol Methods 2000, 237:147-157. 14. Russell SJ, Hawkins RE, Winter G: Retroviral vectors displaying functional antibody fragments. Nucleic Acids Res 1993, 21:1081-1085. 15. Barnett AL, Davey RA, Cunningham JM: Modular organization of the • Friend murine leukemia virus envelope protein underlies the mechanism of infection. Proc Natl Acad Sci USA 2001, 98:4113-4118. This article, which addresses the molecular coupling between receptor binding and fusion activation in the MLV glycoprotein, may elucidate the mechanism of one of the first described retargeted retroviral glycoproteins which was targeted to the erythropoietin receptor (see [8]). 16. Lavillette D, Maurice M, Roche C, Russell SJ, Sitbon M, Cosset F-L: A proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes. J Virol 1998, 72:9955-9965. 17.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Buchholz CJ, Stitz J, Cichutek K: Retroviral cell targeting vectors. Curr Opin Mol Ther 1999, 1:613-621.
2.
Karanavas G, Marin M, Salmons B, Gunzburg WH, Piechaczyk M: Cell targeting by murine retroviral vectors. Crit Rev Oncol Hematol 1998, 28:7-30.
3. Russell SJ, Cosset F-L: Modifying the host range properties of • retroviral vectors. J Gene Med 1999, 1:300-311. A recent exhaustive review on cell targeting by retroviruses. 4.
Benedict CA, Tun RY, Rubinstein DB, Guillaume T, Cannon PM, Anderson WF: Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus–cell fusion. Hum Gene Ther 1999, 10:545-557.
5.
Cosset F-L, Morling FJ, Takeuchi Y, Weiss RA, Collins MKL, Russell SJ: Retroviral retargeting by envelopes expressing an N-terminal binding domain. J Virol 1995, 69:6314-6322.
6.
Zhao Y, Zhu L, Lee S, Li L, Chang E, Soong NW, Douer D, Anderson WF: Identification of the block in targeted retroviralmediated gene transfer. Proc Natl Acad Sci USA 1999, 96:4005-4010.
7.
Sommerfelt MA: Retrovirus receptors. J Gen Virol 1999, 80:3049-3064.
8.
Kasahara N, Dozy AM, Kan YW: Tissue-specific targeting of retroviral vectors through ligand–receptor interactions. Science 1994, 266:1373-1376.
9.
Battini JL, Danos O, Heard JM: Definition of a 14-amino-acid peptide essential for the interaction between the murine leukemia virus amphotropic envelope glycoprotein and its receptor. J Virol 1998, 72:428-435.
10. Valsesia-Wittmann S, Drynda A, Deleage G, Aumailley M, Heard J-M, Danos O, Verdier G, Cosset F-L: Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors. J Virol 1994, 68:4609-4619.
465
Lavillette D, Boson B, Russell S, Cosset F-L: Membrane fusion by murine leukemia viruses is activated in cis or in trans by interactions of the receptor-binding domain with a conserved disulfide loop at the carboxy-terminus of the surface glycoproteins. J Virol 2001, 75:3685-3695.
18. Zhao Y, Lee S, Anderson WF: Functional interactions between monomers of the retroviral envelope protein complex. J Virol 1997, 71:6967-6972. 19. Engelstadter M, Bobkova M, Baier M, Stitz J, Holtkamp N, Chu TH, • Kurth R, Dornburg R, Buchholz CJ, Cichutek K: Targeting human T cells by retroviral vectors displaying antibody domains selected from a phage display library. Hum Gene Ther 2000, 11:293-303. This article describes the selection of antibody fragments from a phage-display library that are able to mediate efficient and potential gene transfer into human T cells. 20. Jiang A, Chu TH, Nocken F, Cichutek K, Dornburg R: Cell-typespecific gene transfer into human cells with retroviral vectors that display single-chain antibodies. J Virol 1998, 72:10148-10156. 21. Hall FL, Gordon EM, Wu L, Zhu NL, Skotzko MJ, Starnes VA, Anderson WF: Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein. Hum Gene Ther 1997, 8:2183-2192. 22. Martin F, Kupsch J, Takeuchi Y, Russell S, Cosset F-L, Collins M: Retroviral vector targeting to melanoma cells by single-chain antibody incorporation in the envelope. Hum Gene Ther 1998, 9:737-746. 23. Hall FL, Liu L, Zhu NL, Stapfer M, Anderson WF, Beart RW, Gordon EM: Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor. Hum Gene Ther 2000, 11:983-993. 24. Liu L, Anderson WF, Beart RW, Gordon EM, Hall FL: Incorporation of tumor vasculature targeting motifs into Moloney murine leukemia virus env escort proteins enhances retrovirus binding and transduction of human endothelial cells. J Virol 2000, 74:5320-5328. 25. Gordon EM, Chen ZH, Liu L, Whitley M, Liu L, Wei D, Groshen S, • Hinton DR, Anderson WF, Beart RW et al.: Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice. Hum Gene Ther 2001, 12:193-204. This study demonstrates that a matrix-targeted retroviral vector inoculated by peripheral vein injection can enhance the delivery of a therapeutic gene in human cancer xenografts implanted in nude mice. 26. Jiang A, Dornburg R: In vivo cell-type-specific gene delivery with retroviral vectors that display single chain antibodies. Gene Ther 1999, 6:1982-1987.
•11. Wu BW, Lu J, Gallaher TK, Anderson WF, Cannon PM: Identification of regions in the Moloney murine leukemia virus SU protein that tolerate the insertion of an integrin-binding peptide. Virology 2000, 269:7-17. A comprehensive study of the properties of retroviruses generated with MLV glycoproteins bearing an insertion of heterologous peptides in various sites of the receptor-binding domain.
27.
12. Kayman SC, Park H, Saxon M, Pinter A: The hypervariable domain • of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system. J Virol 1999, 73:1802-1808. This article demonstrates that peptides and large polypeptides can be displayed at various sites of the proline-rich region of the MLV glycoprotein, without impairing its binding and fusion functions.
28. Peng KW, Pham L, Ye H, Zufferey R, Trono D, Cosset F-L, Russell SJ: •• Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors. Gene Ther 2001, in press. This study addresses the biodistribution of vectors after intravenous inoculation in mice. The results demonstrate that lentiviral vectors carrying tropism-modified glycoproteins can be preferentially retargeted to specific tissues in vivo.
Fielding AK, Maurice M, Morling FJ, Cosset F-L, Russell SJ: Inverse targeting of retroviral vectors: selective gene transfer in a mixed population of hematopoietic and nonhematopoietic cells. Blood 1998, 91:1802-1809.
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Expression vectors and delivery systems
29. Martin F, Neil S, Kupsch J, Maurice M, Cosset F-L, Collins M: Retrovirus targeting by tropism restriction to melanoma cells. J Virol 1999, 73:6923-6929.
42. Rodrigues P, Heard JM: Modulation of phosphate uptake and amphotropic murine leukemia virus entry by posttranslational modifications of PIT-2. J Virol 1999, 73:3789-3799.
30. Peng KW, FJ Morling, Cosset F-L, Murphy G, Russell SJ: A gene delivery system activatable by disease-associated matrix metalloproteinases. Hum Gene Ther 1997, 8:729-738.
43. Skehel JJ, Wiley DC: Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 2000, 69:531-569.
31. Peng KW, Morling FJ, Cosset F-L, Russell SJ: A retroviral gene delivery system activatable by plasmin. Tumor Targeting 1998, 3:112-120.
44. Hatziioannou T, Valsesia-Wittmann S, Russell S, Cosset F-L: Incorporation of fowl plague virus hemagglutinin into murine leukemia virus particles and analysis of the infectivity of the pseudotyped retroviruses. J Virol 1998, 72:5313-5317.
32. Chadwick MP, Morling FJ, Cosset F-L, Russell SJ: Modification of retroviral tropism by display of IGF-I. J Mol Biol 1999, 285:485-494. 33. Peng K-W, Vile RG, Cosset F-L, Russell SJ: Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors. Gene Ther 1999, 6:1552-1557. 34. Morling FJ, Peng K-W, Cosset F-L, Russell SJ: Masking of retroviral envelope functions by oligomerizing peptide adaptors. Virology 1997, 234:51-61. 35. Buchholz CJ, Peng K-W, Morling FJ, Zhang J, Cosset F-L, Russell SJ: In vivo selection of protease cleavage sites from retrovirus display libraries. Nat Biotechnol 1998, 16:951-954. 36. Roux P, Jeanteur P, Piechaczyk M: A versatile approach to the targeting of specific cell types by retroviruses. Proc Natl Acad Sci 1989, 86:9079-9083. 37.
Etienne-Julan M, Roux P, Carillo S, Jeanteur P, Piechaczyk M: The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker. J Gen Virol 1992, 73:3251-3255.
38. Snitkovsky S, Young JA: Cell-specific viral targeting mediated by a soluble retroviral receptor–ligand fusion protein. Proc Natl Acad Sci USA 1998, 95:7063-7068. 39. Snitkovsky S, Niederman TM, Carter BS, Mulligan RC, Young JA: A TVA-single-chain antibody fusion protein mediates specific targeting of a subgroup A avian leukosis virus vector to cells expressing a tumor-specific form of epidermal growth factor receptor. J Virol 2000, 74:9540-9545. 40. Boerger AL, Snitkovsky S, Young JA: Retroviral vectors preloaded • with a viral receptor–ligand bridge protein are targeted to specific cell types. Proc Natl Acad Sci USA 1999, 96:9867-9872. This study demonstrates that it is possible to retarget avian vectors to specific cell types through the use of a retroviral receptor–ligand fusion protein preloaded on the vector particles. 41. Kizhatil K, Albritton LM: Requirements for different components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion. J Virol 1997, 71:7145-7156.
45. Lin AH, Kasahara N, Wu W, Stripecke R, Empig CL, Anderson WF, Cannon PM: Receptor-specific targeting mediated by the coexpression of a targeted murine leukemia virus envelope protein and a binding-defective influenza hemagglutinin protein. Hum Gene Ther 2001, 12:323-332. 46. Hatziioannou T, Delahaye E, Martin F, Russell SJ, Cosset F-L: • Retroviral display of functional binding domains fused to the amino-terminus of influenza haemagglutinin. Hum Gene Ther 1999, 10:1533-1544. This article establishes that it is possible to engineer the influenza virus hemagglutinin protein by fusing ligands to its N-terminal end without affecting its fusion activity. It was then possible to use such chimeric glycoproteins to target retroviral vectors to specific cell-surface receptors. 47.
Patterson SM, Swainsbury R, Routledge EG: Antigen-specific membrane fusion mediated by the haemagglutinin protein of influenza A virus: separation of attachment and fusion functions on different molecules. Gene Ther 1999, 6:694-702.
48. Christodoulopoulos I, Cannon P: Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors. J Virol 2001, 75:4129-4138. 49. Boson B, Sandrin V, Nègre D, Cosset F-L: Efficient transduction of primary primate PBLs with SIV-derived lentiviral vectors pseudotyped with a cytoplasmic tail-modified glycoprotein derived from RD114. J Gen Med 2001, in press. 50. Morizono K, Bristol G, Xie Y-M, Kung SK-P, Chen ISC: Antibodydirected targeting of retroviral vectors via cell surface antigens. J Virol 2001, 75:8016-8020. 51. Pizzato M, Marlow SA, Blair ED, Takeuchi Y: Initial binding of murine leukemia virus particles to cells does not require specific Env–receptor interaction. J Virol 1999, in press. 52. Pizzato M, Blair ED, Fling M, Kopf J, Tomassetti A, Weiss RA, Takeuchi Y: Evidence for non-specific adsorption of targeted retrovirus vector particles to cells. Gene Ther 2001, in press. 53. Doms RW, Trono D: The plasma membrane as a combat zone in the HIV battlefield. Genes Dev 2000, 14:2677-2688.