Targeting Retroviral Vectors

Efforts to target retroviral vectors to specific cell types have concentrated largely on engineering natural retroviral envelope proteins, and in particular the rodent cell-specific eco-tropic Moloney MuLV protein [reviewed in (44,45)]. Ret-roviral Env proteins exist as an oligomeric complex (in the case of MuLV Env, probably a trimer), comprising 2 subunits, the surface (SU) protein that contains the receptor recognition domain and the transmembrane (TM) protein that anchors the complex in the retroviral envelope (Fig. 6A). The binding of SU to a specific cell surface receptor is believed to trigger conformational changes in SU and the associated TM protein that result in the exposure of a hydrophobic stretch of amino acids at the N-terminus of TM, the fusion peptide, and the subsequent fusion between viral and host cell membranes (Fig. 6B). The challenge of engineering Env has been to redirect

Figure 6 Retroviral Env protein. (A) The Env protein consists of 2 noncovalently linked subunits, the SU protein, which contains the residues that interact with the receptor, and the membrane-anchored TM protein, which promotes the fusion of viral and host cell membranes. At the N-terminus of TM is a stretch of hydrophobic amino acids called the fusion peptide. (B) The retroviral Env protein is oligomeric and MuLV Env probably exists as a trimer. Following binding to its receptor, the Env complex is believed to undergo conformational changes that result in the exposure of the fusion peptide, enabling it to interact with the host cell membrane and initiate the fusion process.

Figure 6 Retroviral Env protein. (A) The Env protein consists of 2 noncovalently linked subunits, the SU protein, which contains the residues that interact with the receptor, and the membrane-anchored TM protein, which promotes the fusion of viral and host cell membranes. At the N-terminus of TM is a stretch of hydrophobic amino acids called the fusion peptide. (B) The retroviral Env protein is oligomeric and MuLV Env probably exists as a trimer. Following binding to its receptor, the Env complex is believed to undergo conformational changes that result in the exposure of the fusion peptide, enabling it to interact with the host cell membrane and initiate the fusion process.

binding of the SU moiety to a heterologous cell surface molecule, while retaining the ability of such an interaction to recapitulate the natural postbinding events that lead to fusion. This has proved a daunting task and, despite early optimism in the field, there have now been numerous reports of failure of targeting strategies with MuLV Env proteins (45-48). Even the few reported success stories appear to be ligand speicifc (49), and a robust and general strategy for targeting MuLV Env proteins has not been forthcoming. The Env protein of spleen necrosis virus may be more amenable to engineering (50,51), although success here also appears to be ligand specific (52)

Two broad approaches have been taken to produce targeted envelope proteins (Fig. 7). First, the natural receptor-binding domain of the SU protein can be replaced with a ligand or single-chain antibody designed to bind to a specific cell surface molecule on the target cell. A whole range of receptors have been targeted in this way, but the difficulty remains that even when specific binding can be obtained between the engineered vector and the target cell receptor, the subsequent fusion event is not triggered and gene transfer is correspondingly low (46,48). It is apparent that engineering the receptor-binding domain of SU to redirect binding while maintaining the ability of the envelope protein to carry out fusion will require a better understanding of the structure-function relationships within the envelope protein complex. The 3-dimensional structure of the receptor-binding domain of the murine eco-tropic (Friend strain) SU protein is available (53), and several structure-function studies have delineated various functional domains within MuLV Env that shed light on the pathway of

Figure 7 Targeted Env proteins. (A) Wild-type Env protein. (B) Targeting to heterologous receptors: the region of SU that interacts with the natural receptor is replaced by a heterologous binding ligand, such as a small peptide or scFv insert. (C, D) Tethering strategies: the natural receptor-binding interaction is retained, but the vectors are first concentrated on target cells by the action of the additional targeting ligand. In some cases (D), the targeting ligand obscures the natural receptor binding site, which is only revealed after the binding ligand has been removed, for example, by proteolytic cleavage. (E) Bridging strategy: a chimeric protein comprising a soluble receptor and a targeting ligand binds the vector to target cells. The soluble receptor activates the Env protein, allowing fusion to occur with a cell that does not express the natural receptor. (F) Coexpression strategy: receptor binding and fusion functions are separated between 2 different molecules. For example, target cell binding is directed by a chimeric MuLV Env and the fusion component is provided by a binding-defective but fusion-competent influenza HA protein.

signal transmission within the Env protein complex (54-57). Hopefully, such information will allow a more rational approach to engineering retroviral Env proteins in the future.

In a second broad approach to Env targeting that could be called ''tethering,'' the interaction with the native receptor is maintained so that entry occurs through the natural route, but the vectors are concentrated on certain cell types or at certain sites by the presence of an additional binding moiety. The insertion of a collagen-binding ligand into the ecotropic MuLV Env protein, for example, did not perturb the ability of the protein to transduce rodent cells but did allow an effective concentration of the vector at sites of collagen deposition (58). More recently, incorporating this targeting motif into ampho-tropic (human cell tropic) MuLv Env proteins has resulted in enhanced gene delivery to sites of wound healing following balloon angioplasty, and allowed in vivo gene delivery to human cancer xenografts in nude mice following systemic administration (59).

A different strategy to concentrate vectors on cells expressing the epidermal growth factor (EGF) receptor has been re ported that uses a chimeric MuLV Env protein where the natural receptor-binding site is initially blocked by an EGF moiety (60). Following binding to the EGF receptor-expressing cells, the EGF ligand is removed by the action of a protease (envisioned to be present on the cell surface and which can therefore be made cell specific). This cleavage event frees the Env protein to interact with its natural receptor and to subsequently enter the cells. Such a strategy seems particularly suited to cancer cell targeting.

Recently, ''bridging constructs'' have been described for the Env protein from avian leukosis virus (ALV) that combine an EGF targeting domain with the extracellular domain of the ALV receptor (61). These hybrid proteins can be preloaded onto ALV Env pseudotyped vectors and can subsequently ''bridge'' them to target cells expressing the EGF receptor (62). The interaction of the receptor fragment with Env protein triggers the normal fusion process, thereby allowing entry of the vector to the EGF receptor-expressing target cells in the absence of the natural receptor. The success of this system appears to be the result of 2 fortuitous properties of the ALV entry process: (1) the receptor is a single membrane-spanning protein that is amenable to engineering as a soluble domain, and (2) the ALV Env is triggered to induce virus-cell fusion in a 2-stage process, requiring both receptor binding to prime the Env and low pH exposure during endocytosis to complete the process (63).

Finally, in a strategy mimicking the 2-protein approach used by several enveloped viruses, notably the paramyxovi-ruses, it is possible to combine 2 different moieties on a ret-roviral vector particle—a targeting protein to bind the vectors to specific cells and a fusion protein that will then promote entry (64). Using a modified influenza hemagglutinin (HA) protein that is no longer capable of binding to its native receptor, the ubiquitously expressed sialic acid (65), we have demonstrated enhanced entry into Flt-3-expressing cells when this HA is coexpressed with a binding-competent but fusion-defective MuLV Env containing the Flt-3 ligand (66).

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