Abstract
Lentiviral vectors (LVs) are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV) glycoproteins, the hemagglutinin (H), responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv) specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV) in presence of α-MV antibody-positive human plasma. At plasma dilution 1∶160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1∶80 an at least 4-times higher multiplicity of infection (MOI) of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against α-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of α-MV antibodies interfering with entry via the natural MV receptors. These results are promising for in vivo applications of targeting vectors in humans.
Highlights
For genetic modification of primary cells, lentiviral vectors (LVs) are the most suitable vectors as they stably integrate the transferred gene into the genome of dividing as well as nonproliferating cells [1]
The ability to transduce target cells in presence of a-measles virus (MV) antibody-positive human plasma was investigated for targeting vectors specific to human CD20, CD105 or CD133 (Fig. 1) as well as for non-targeting vectors, pseudotyped with the MV glycoproteins derived from the NSe variant of the MV vaccine strain Edmonston B (MVNSe-LV) or the MV wild-type strain IC-B (MVwt-LV, Fig. 1)
Targeting vectors as well as MVNSe-LV, MVwt-LV and VSVGLV vectors, which transfer the genetic information for the enhanced green fluorescent protein (EGFP), were incubated in serial complement inactivated a-MV antibody-positive human plasma dilutions, before the mixtures were added to the respective CD20, CD105- or CD133-positive target cells
Summary
For genetic modification of primary cells, lentiviral vectors (LVs) are the most suitable vectors as they stably integrate the transferred gene into the genome of dividing as well as nonproliferating cells [1]. The lentiviral envelope protein can be exchanged with glycoproteins derived from other viruses (pseudotyping) and current state-of-the-art vectors are equipped with the glycoprotein of vesicular stomatitis virus (VSVG), which is very stable and allows production of vectors with high titers It has a wide tropism and mediates nonselective cell entry into basically all types of mouse, rat and human cells. The desired receptor specificity is provided by displaying a singlechain antibody (scFv) specific for the target receptor on the mutated H protein (Hmut-scFv) This way, very different cell surface molecules including type1-membrane glycoproteins (CD105), pentaspan membrane glycoproteins (CD133), membrane tetraspan calcium channels (CD20) as well as multi-subunit ion-channels (glutamate receptors, GluR) can be used for entry by these vectors. Rapid clearance of targeting vectors by neutralizing a-H antibodies in the plasma may impair in vivo applications in humans
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