A Novel Approach and a Novel Mechanism for Stealthing Gene Delivery Vehicles
Authors: Thomas Wickham
Journal: Molecular Therapy (2000)

One of the “holy grails” for gene delivery is a vehicle that can be injected systemically but will then deliver genes to a diseased tissue of interest. A major obstacle to this goal for at least some vehicles is the negative effect of blood protein components. These components can rapidly bind to the vehicle, interfere with efficient gene transfer to the target tissue, and mediate rapid clearance of the vehicle from the circulation. The resultant shortened half-life of the vehicles in the bloodstream prevents adequate amounts from reaching the target tissue. The term “opsonization” is used to describe the innate immune process by which serum components bind to foreign matter (such as pathogens or particulates) or unwanted matter (such as cell debris) in the bloodstream. Opsonization usually leads to inactivation through the complement system and/or clearance by macrophages in the liver. In the case of some liposomal gene delivery vehicles, opsonization is well-documented and is generally recognized to affect the biodistribution properties and/or efficacy of these vehicles. In this issue of Molecular Therapy, Sokoloff et al. describe an important step forward in understanding the mechanisms by which one class of drug and gene delivery vehicles could be inactivated and/or cleared by opsonization (1Sokoloff A.V. Bock I. Zhang G. Sebestyén M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Perhaps even more importantly, the authors also describe a separate, but related, “antiopsonic” mechanism by which the inactivation of their model vehicle, phage T7, by complement-mediated inactivation can be avoided. This mechanism has implications for the design of improved gene transfer vehicles for which opsonization could prevent efficient gene delivery. In their work, Sokoloff et al. employed the T7 phage displaying random peptides at the carboxy-terminus of the 10B phage coat protein as a model for gene delivery vehicles containing peptide ligands. Surprisingly, they found that peptide-tagged T7 phage, but not the non-tagged phage, were inactivated in serum by the complement system and correspondingly had short survival times in the circulation. This result indicates that the inactivation is specific to the peptide tag. Interestingly, the group found that natural IgM antibodies (whose presence in the blood did not require preimmunization with corresponding antigens) mediated the inactivation. IgM binding to the peptide tag led to inactivation of the phage through the classical complement pathway in mouse, rat, and human serum. Their data also suggest that the neutralization by the IgM antibodies was not due to a single class of antibody neutralizing all the different peptide tags, but to natural IgM antibodies specific to the peptide C-terminal sequences. In this way, the researchers identified a mechanism by which delivery vehicles containing C-terminal peptide tags can be inactivated by complement. In addition to identifying a new inactivation mechanism for delivery vehicles containing C-terminal peptide tags, Sokoloff et al. developed an approach to circumvent the problem, an approach that has potentially broader application to a larger class of vehicles. Although the majority of peptide tags led to inactivation, the T7 peptide display library could also be used to select for peptides in either rat or human serum that allowed the phage to avoid complement-mediated inactivation as well as to increase phage survival and half-life in the blood of macrophage-depleted rats. In rat serum, phage with increased survival were found to have peptides containing a C-terminal lysine or arginine. These phage bound to the serum protective factor (CRP) and were protected from complement-mediated destruction. Interestingly, the same phage were not protected in human serum. However, by using the same type of selection strategy for phage survival in human serum, peptides that contained tyrosine residues were able to protect the phage. In human serum, it appeared that the protective effect was endowed through binding of the tyrosine-containing peptide to α-macroglobulin. Therefore, these results suggest a novel approach to protect certain gene delivery vehicles by “stealthing” them with peptides that bind “antiopsonic” blood components. This mechanism is markedly different from other approaches, such as PEGylation, which seek to protect a vehicle from recognition by any blood component. Interestingly, the stealthing mechanism identified with peptides is not completely novel in the sense that many bacterial pathogens have previously been shown to use similar types of mechanisms to evade innate immunity. Certainly, the actual mechanism described for the complement activation and clearance of the peptide-tagged T7 phage may have relevance only for those gene transfer vehicles in which a C-terminal peptide is exposed on the vehicle. For example, this exact mechanism is obviously not involved in the clearance from the bloodstream of liposomes that do not contain peptide tags. However, IgM binding to liposomes that do not contain peptides has been observed, which could indicate broader binding characteristics of natural IgM antibodies. In addition, complement-mediated inactivation is unlikely to occur for other viral vehicles, such as adenovirus or adeno-associated virus. For example, adenovirus vectors are not inactivated by complement-mediated mechanisms, as shown by their high stability in naive serum that does not contain neutralizing antibodies. Although adenovirus vectors are not directly inactivated due to opsonization, it still is unknown if opsonization by serum plays a role in the rapid clearance of adenovirus vectors and other viral vectors from the bloodstream by the liver. Recent preliminary evidence suggests that adenovirus vectors are rapidly taken up by Kupffer cells in the liver; however, it is unknown whether this uptake is mediated by the known interactions of adenovirus with its native receptors (CAR and integrins) or whether the uptake is mediated by other interactions, such as opsonization of the vector. For enveloped viruses, the evidence may be a little clearer as opsonization of sugar residues on the envelope proteins of influenza virus by a class of opsonins called “collectins” has been shown to mediate clearance by the liver. The use of peptide phage display to select for a desired characteristic, namely increased serum stability, powerfully demonstrates the advantage of selection techniques. Moreover, while the IgM-mediated inactivation mechanism described for peptide-tagged T7 may apply only to those gene transfer vehicles containing exposed C-terminal peptides, the combined separate mechanism and selective approach by which the inactivation can be avoided is potentially a new way to protect gene therapy vehicles, viral or nonviral, against rapid clearance and/or inactivation in vivo. In theory, the peptides identified that could stealth the peptide-tagged T7 phage may be applicable to diverse gene transfer vehicles where opsonization leads to inactivation or clearance.