Abstract

Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.

Highlights

  • Over the past two and a half decades, lentiviral (LV) vectors have emerged as powerful tools for transgene delivery [1]

  • The scaffold RNA was in turn base-paired to a specific short RNA sequence, such as an RNA aptamer

  • Our results showed that the exposure of the A431 cells to the J18 aptamer in the presence of recombinant EGF (rEGF) or recombinant EGFR (rEGFR) resulted in diminished flow cytometry signals (Figure 3A, left and right panels, respectively)

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Summary

Introduction

Over the past two and a half decades, lentiviral (LV) vectors have emerged as powerful tools for transgene delivery [1]. A non-primate LV vector system based on equine infectious anemia virus (EIAV) has been investigated clinically to treat ocular disorders [8]. In another in vivo application, an HIV-1-based, integrationdeficient LV vector expressing the NY-ESO-1 cancer testis antigen targeted to dendritic cells was used to promote an immune response against NY-ESO-1-expressing tumors [9]

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