Protein transduction by pseudotyped lentivirus-like nanoparticles

Abstract

A simple, efficient and reproducible method to transduce proteins into mammalian cells has not been established. Here we describe a novel protein transduction method based on a lentiviral vector. We have developed a method to package several thousand foreign protein molecules into a lentivirus-like nanoparticle (LENA) and deliver them into mammalian cells. In this proof-of-concept study, we used b-lactamase (BlaM) as a reporter molecule. The amino-terminus of BlaM was fused to the myristoylation signal of lyn, which was placed upstream of the amino-terminus of Gag (BlaM-gag-pol). By co-transfection of plasmids encoding BlaM-gag-pol and vesicular stomatitis virus-G (VSV-G) into 293T cells, LENA were produced containing BlaM enzyme molecules as many as Gag per capsid, which has been reported to be B5000 molecules, but lacking the viral genome. Infection of 293T and MT-4 cells by VSV-G-pseudotyped BlaM-containing LENA led to successful transduction of BlaM molecules into the cell cytoplasm, as detected by cleavage of the fluorescent BlaM substrate CCF2-AM. LENA-mediated transient protein transduction does not damage cellular DNA, and the preparation of highly purified protein is not necessary. This technology is potentially useful in various basic and clinical applications. Gene Therapy (2011) 18, 936-941;

Retrovirus-based vectors for transient and permanent cell modification

“…However, when embedding a foreign protein into Gag, examples of MLV-based RPT describe the need to trans-complement wild-type Gag-Pol during packaging [75 ], thereby reducing the amount of foreign proteins that can be co-delivered. This requirement was overcome in the LV context by equipping a POI fused amino-terminally to Gag with a heterologous myristoylation signal for efficient membrane targeting, which is required for packaging into nascent particles [77]. This strategy had previously been shown to also increase the yield for iLVV production [82].…”

Delivering the Goods for Genome Engineering and Editing

“…Studies dating back to the 102 In the context of lentiviral particles, work by the group of Jun Komano has been instrumental in showing the robust delivery of proteins, including β-lactamase and caspase 3, fused to the matrix protein at the N-terminus of Gag and GagPol. 103,104 Furthermore, foreign proteins fused to the p6 protein at the C-terminal end of Gag 105-108 and inserted between the matrix and capsid proteins 109 have been successfully incorporated in lentiviral particles.…”

“…47 A schematic representation of this approach is provided in Figure 2. Using the packaging construct design developed by Aoki et al, 103 we fused hyPBase to the N-terminal end of Gag and separated the two domains with a HIV-1 protease cleavage motif. Also, Gag was modified by adding an N-terminal myristoylation signal derived from the Lyn kinase.…”

The Use of Nanostructures for DNA Transfection

“…In general, the genetic information required for the natural replicative cycle of the virus is removed from the viral genome and replaced by the genes of interest [47]. Although viral transfection has a better efficiency in DNA delivery to the target cell, there are still several problems in its use such as high cytotoxicity, the high cost, low reproducibility and concerns about biosafety [2].…”