Targeted in vivo transduction, entailing direct administration of viral vector preparations to patients, is the next big step in gene therapy. To redirect lentiviral vector (LV) particles selectively to desired cell type(s), different glycoproteins have been engineered to alter their tropism, including the glycoprotein G from Vesicular Stomatitis Virus (VSV-G) as the most commonly employed tropism-defining protein for LV particles. For detargeting from its natural receptor, the low-density lipoprotein receptor (LDLR), a VSV-G variant with two blinding substitutions, K47Q and R354A, is commonly used (VSV-G.pub). We provide first evidence for insufficient blinding of VSV-G.pub in human and murine cell lines and primary cells. In silico modeling pointed toward only slightly reduced LDLR binding affinity of VSV-G.pub. To lower the affinity further, we generated three novel VSV-G variants based on charge-reversing substitutions in two, four, or six key residues. These variants achieved a more stringent blinding compared with VSV-G.pub in the tested cell lines and primary cells. Codisplay of a CD4 binder enabled lentiviral particles pseudotyped with the novel variants to selectively transduce CD4-expressing cells. In summary, we present improved VSV-G variants with a better on/off-target ratio as attractive tools for in vivo gene therapy applications.
Felix L. Warnecke, M. Ertelt, Anjali Shrivastava et al.· Human Gene Therapy· 0 citations
Lentiviral vectors have revolutionized gene therapy by efficient and stable transduction of dividing and non-dividing cells, their large packaging capacity, and their compatibility with pseudotyping to alter viral tropism. The vesicular stomatitis virus glycoprotein (VSV-G) is widely used as a viral envelope protein of choice to pseudotype lentiviral vector particles as it confers exceptional particle stability and a broad tropism, due to the ubiquitous nature of the low-density lipoprotein receptor (LDLR). While this broad tropism facilitates transduction of diverse cell types, it precludes accurate in vivo targeting of specific cell populations. Structural insights into VSV-G have made receptor-blinding possible and revealed sites amenable to mutation while preserving fusion capacity. Coupled with targeting moieties, VSV-G pseudotyped lentiviral particles are redirected towards cells expressing target antigens. Such targeted vectors open new possibilities for in vivo gene therapy across oncology, infectious diseases, transplantation medicine, and other diseases. Use of targeted vectors will make in vivo gene therapy more accessible than cost-intensive ex vivo gene therapies. Since targeted vectors will be available as 'off-the-shelf' drugs, they will also drastically reduce time-to-treatment. This review highlights advances in bioengineering to exploit the versatility of VSV-G-pseudotyped lentiviral vectors and explores their vast potential for targeted gene delivery.
Anjali Shrivastava, Felix L. Warnecke, J. Schott et al.· Molecular Therapy· 0 citations