On Target: New Envelopes for Lentiviral Vectors

Abstract

Every moment of our existence is based on the peaceful and coordinated interplay of thousands of different types of cells in our body. However, this enjoyable complexity can pose a major obstacle for targeted treatment interventions, as diseases often result from the malfunction, deletion, or expansion of well-defined subsets of cells that may not be easily singled out in the human body. Targeting the relevant cell type thus represents a great challenge for molecular therapy, with major consequences for both efficiency and biosafety.As the various families of viral gene vectors show substantial differences in their mechanisms of particle assembly and host-cell interactions, specific solutions must be developed for each vector type to achieve efficient cell targeting. In the case of lentiviral vectors, which continue to emerge as one of the more promising viral gene delivery systems, various approaches have been introduced to modify the molecules through which they recognize their target cells and initiate the process of particle uptake. These molecules are known as the retroviral envelope proteins, typically simple glycoproteins comprising a transmembrane domain and a surface domain, generated from a single precursor protein by post-translational proteolytic cleavage and held together by disulfide bridges. The parent HIV-1 virus demonstrates the power of this principle for cell-specific targeting, as its envelope precisely guides entry into cells that express the CD4 receptor and chemokine co-receptors CXCR4 or CCR5.Several attempts have been made to expand or retarget the host range of lentiviral vector particles with envelope proteins derived from other retroviruses or enveloped RNA viruses, the most widely used strategy still being “pseudotyping” with the glycoprotein of vesicular stomatitis virus. Thus, potent vector preparations could be generated that mediate almost ubiquitous cell entry,1Dull T Zufferey R Kelly M Mandel RJ Nguyen M Trono D et al.A third-generation lentivirus vector with a conditional packaging system.J Virol. 2008; 72: 8463-8471Google Scholar broad tropism coupled with potential activation of specific cell types by co-display of cytokine domains,2Verhoeyen E Dardalhon V Ducrey-Rundquist O Trono D Taylor N Cosset FL IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes.Blood. 2003; 101: 2167-2174Crossref PubMed Scopus (99) Google Scholar or greatly increased tropism for certain cell types in a given disease context.3Miletic H Fischer YH Neumann H Hans V Stenzel W Giroglou T et al.Selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins.Hum Gene Ther. 2004; 15: 1091-1100Crossref PubMed Scopus (46) Google Scholar The recently introduced technology based on Sindbis glycoproteins allows lentiviral vector pseudotyping with engineered envelope proteins containing designer domains for cell entry.4Yang L Bailey L Baltimore D Wang P Targeting lentiviral vectors to specific cell types in vivo.Proc Natl Acad Sci USA. 2006; 103: 11479-11484Crossref PubMed Scopus (128) Google Scholar None of the above principles, however, has been shown to be versatile and potent enough to mediate rational and predictable retargeting using an uptake mechanism that is based on direct fusion of lentiviral particles with their target cells of interest, thus avoiding the potentially severe restriction of infectivity caused by endosomal uptake.In this issue of Molecular Therapy, Christian Buchholz's group introduces a new concept for lentiviral vector pseudotyping that utilizes basic components of the surface proteins of measles virus.5Funke S Maisner A Mühlebach MD Koehl U Grez M Cattaneo R et al.Targeted cell enty of lentiviral vectors.Mol Ther. 2008; 16: 1427-1436Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar Consisting of a “specialized fuser”—the fusion (F) protein, and the H protein, which combines fusion helper activity with a distinct, simply organized cell recognition function, the measles surface proteins are excellent candidates for directed targeting of enveloped viral vector particles, as previously demonstrated in the context of oncolytic measles variants.6Nakamura T Peng KW Harvey M Greiner S Lorimer IA James CD et al.Rescue and propagation of fully retargeted oncolytic measles viruses.Nat Biotechnol. 2005; 23: 209-214Crossref PubMed Scopus (220) Google Scholar Buchholz and colleagues managed to identify variants of F and H proteins that are not only efficiently incorporated into lentiviral vector particles but that also retain fusion activity. The researchers furthermore defined an optimal ratio of coexpression of the two proteins for lentiviral pseudotyping. They present a convincing paradigm of the potential of this elegant principle by targeting lentiviral vectors into resting B cells, a population that previously resisted efficient genetic engineering attempts using lentiviral vectors. In well-designed cell-mixing experiments, they demonstrated that the re-engineered H proteins containing an epitope-recognition domain of a monoclonal antibody directed against the CD20 protein expressed on B cells mediate not only efficient but also specific gene transfer into the target of interest.The beauty of the new, “morbilliform” targeting principle is the versatility of the H protein in incorporating targeting domains derived from single-chain antibodies—and potentially from many other ligands. Although Buchholz and colleagues did not demonstrate the power of their targeting approach with a great number of different cell types or in a disease model, it can be expected that the F–H protein couple will find widespread use in targeting lentiviral particles to the cells of interest.What remains to be addressed is the potential of this system to de-target particles from unspecific trapping events, an issue that is of great relevance for in vivo delivery of particles. Also to be determined is the utility of this principle for the generation of stable packaging cells, which continues to be the most important technical hurdle to the successful development of lentiviral vectors beyond phase I clinical trials. Of note, the separation of the envelope protein into two distinct genes should further reduce the potential for the generation of replication-competent lentivirus in packaging cells. Finally, it will be of interest to see whether lentiviral vectors other than HIV-1 or other types of retroviral vectors can be pseudotyped using the same principle. If initial attempts are not successful, the systematic approach described by Buchholz and colleagues to identify suitable variants of H and F proteins for particle incorporation may well guide the successful adaptation of this promising targeting principle to many other types of vectors. Every moment of our existence is based on the peaceful and coordinated interplay of thousands of different types of cells in our body. However, this enjoyable complexity can pose a major obstacle for targeted treatment interventions, as diseases often result from the malfunction, deletion, or expansion of well-defined subsets of cells that may not be easily singled out in the human body. Targeting the relevant cell type thus represents a great challenge for molecular therapy, with major consequences for both efficiency and biosafety. As the various families of viral gene vectors show substantial differences in their mechanisms of particle assembly and host-cell interactions, specific solutions must be developed for each vector type to achieve efficient cell targeting. In the case of lentiviral vectors, which continue to emerge as one of the more promising viral gene delivery systems, various approaches have been introduced to modify the molecules through which they recognize their target cells and initiate the process of particle uptake. These molecules are known as the retroviral envelope proteins, typically simple glycoproteins comprising a transmembrane domain and a surface domain, generated from a single precursor protein by post-translational proteolytic cleavage and held together by disulfide bridges. The parent HIV-1 virus demonstrates the power of this principle for cell-specific targeting, as its envelope precisely guides entry into cells that express the CD4 receptor and chemokine co-receptors CXCR4 or CCR5. Several attempts have been made to expand or retarget the host range of lentiviral vector particles with envelope proteins derived from other retroviruses or enveloped RNA viruses, the most widely used strategy still being “pseudotyping” with the glycoprotein of vesicular stomatitis virus. Thus, potent vector preparations could be generated that mediate almost ubiquitous cell entry,1Dull T Zufferey R Kelly M Mandel RJ Nguyen M Trono D et al.A third-generation lentivirus vector with a conditional packaging system.J Virol. 2008; 72: 8463-8471Google Scholar broad tropism coupled with potential activation of specific cell types by co-display of cytokine domains,2Verhoeyen E Dardalhon V Ducrey-Rundquist O Trono D Taylor N Cosset FL IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes.Blood. 2003; 101: 2167-2174Crossref PubMed Scopus (99) Google Scholar or greatly increased tropism for certain cell types in a given disease context.3Miletic H Fischer YH Neumann H Hans V Stenzel W Giroglou T et al.Selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins.Hum Gene Ther. 2004; 15: 1091-1100Crossref PubMed Scopus (46) Google Scholar The recently introduced technology based on Sindbis glycoproteins allows lentiviral vector pseudotyping with engineered envelope proteins containing designer domains for cell entry.4Yang L Bailey L Baltimore D Wang P Targeting lentiviral vectors to specific cell types in vivo.Proc Natl Acad Sci USA. 2006; 103: 11479-11484Crossref PubMed Scopus (128) Google Scholar None of the above principles, however, has been shown to be versatile and potent enough to mediate rational and predictable retargeting using an uptake mechanism that is based on direct fusion of lentiviral particles with their target cells of interest, thus avoiding the potentially severe restriction of infectivity caused by endosomal uptake. In this issue of Molecular Therapy, Christian Buchholz's group introduces a new concept for lentiviral vector pseudotyping that utilizes basic components of the surface proteins of measles virus.5Funke S Maisner A Mühlebach MD Koehl U Grez M Cattaneo R et al.Targeted cell enty of lentiviral vectors.Mol Ther. 2008; 16: 1427-1436Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar Consisting of a “specialized fuser”—the fusion (F) protein, and the H protein, which combines fusion helper activity with a distinct, simply organized cell recognition function, the measles surface proteins are excellent candidates for directed targeting of enveloped viral vector particles, as previously demonstrated in the context of oncolytic measles variants.6Nakamura T Peng KW Harvey M Greiner S Lorimer IA James CD et al.Rescue and propagation of fully retargeted oncolytic measles viruses.Nat Biotechnol. 2005; 23: 209-214Crossref PubMed Scopus (220) Google Scholar Buchholz and colleagues managed to identify variants of F and H proteins that are not only efficiently incorporated into lentiviral vector particles but that also retain fusion activity. The researchers furthermore defined an optimal ratio of coexpression of the two proteins for lentiviral pseudotyping. They present a convincing paradigm of the potential of this elegant principle by targeting lentiviral vectors into resting B cells, a population that previously resisted efficient genetic engineering attempts using lentiviral vectors. In well-designed cell-mixing experiments, they demonstrated that the re-engineered H proteins containing an epitope-recognition domain of a monoclonal antibody directed against the CD20 protein expressed on B cells mediate not only efficient but also specific gene transfer into the target of interest. The beauty of the new, “morbilliform” targeting principle is the versatility of the H protein in incorporating targeting domains derived from single-chain antibodies—and potentially from many other ligands. Although Buchholz and colleagues did not demonstrate the power of their targeting approach with a great number of different cell types or in a disease model, it can be expected that the F–H protein couple will find widespread use in targeting lentiviral particles to the cells of interest. What remains to be addressed is the potential of this system to de-target particles from unspecific trapping events, an issue that is of great relevance for in vivo delivery of particles. Also to be determined is the utility of this principle for the generation of stable packaging cells, which continues to be the most important technical hurdle to the successful development of lentiviral vectors beyond phase I clinical trials. Of note, the separation of the envelope protein into two distinct genes should further reduce the potential for the generation of replication-competent lentivirus in packaging cells. Finally, it will be of interest to see whether lentiviral vectors other than HIV-1 or other types of retroviral vectors can be pseudotyped using the same principle. If initial attempts are not successful, the systematic approach described by Buchholz and colleagues to identify suitable variants of H and F proteins for particle incorporation may well guide the successful adaptation of this promising targeting principle to many other types of vectors.