MLV based viral-like-particles for delivery of toxic proteins and nuclear transcription factors

Abstract

We have developed nanoparticles based on Murine Leukemia Virus virus-like-particles (VLPs) that efficiently deliver therapeutic bioactive proteins in their native state into target cells. Nuclear transcription factors and toxic proteins were incorporated into the VLPs from stable producer cells without transducing viral-encoded genetic material. Delivery of nuclear transcription factors required incorporation of nuclear export signals (NESs) into the vector backbone for the efficient formation of VLPs. In the presence of an appropriate targeting Env glycoprotein, transcription factors delivered and activated nuclear transcription in the target cells. Additionally, we show delivery of the bacterial toxin, MazF, which is an ACA-specific mRNA interferase resulted in the induction of cell death. The stable producer cells are protected from the toxin through co-expression of the anti-toxin MazE and continuously released MazF incorporating VLPs. This highly adaptable platform can be harnessed to alter and regulate cellular processes by bioactive protein delivery.

Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection

“…The levels of late reverse transcription products and two one-long-terminal-repeat (2-LTR) circles of MLV in infected NIH3T3 cells were quantified by real-time quantitative PCR analysis using iQ™ SYBR® Green Supermix (BIO-RAD) followed protocol described in (Wu and Roth, 2014). Genomic DNA (50 ng) from MLV-infected NIH3T3 cells was used as the template for the detection of MLV late reverse transcription products and cellular GAPDH, and 250 ng of genomic DNA was used for the detection of MLV 2-LTR circles.…”

Retrovirus-based vectors for transient and permanent cell modification

“…Vice versa, the presence of specific subcellular localization signals in POI has been revealed to occasionally impact RPT particle packaging. In a recent approach to transfer the reprogramming TFs Oct4, Sox2, Klf4 and c-Myc by GV RPT, nuclear localization signals contained within the TFs led to retention of Gag fusion proteins within the nucleus, thereby interfering with efficient particle production [83]. This obstacle could be solved based on the concept of PR recognition sites.…”

“…Additionally inserted nuclear export signals (NES) were employed to relieve nuclear retention and thereby allow membrane targeting and packaging of the Gag-fusions. As NES after target cell entry would probably have interfered with proper localization and thus function of the TFs, PR target sites served to remove these signals after packaging [83].…”

Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins

“…In FeLV-A, the VRA and VRB each include a pair of cysteines, which have the potential to form intramolecular disulfide bonds (5). The cysteine contents of MLV and FeLV-B SUs are more complex; ecotropic MLV SU has six cysteines in the VRA and two in the VRB (2, 13), while amphotropic MLV and FeLV B SUs have four cysteines in both VRA and VRB (1,2,14,15).Targeted entry of retroviral particles is a key interest in gene and protein delivery (16,17). Insertion of ligand binding domains, single-chain antibody (Ab) regions, and other conjugates into the Env SU of MLV and FeLV has been explored, with relative success (reviewed in reference 16).…”

“…Targeted entry of retroviral particles is a key interest in gene and protein delivery (16,17). Insertion of ligand binding domains, single-chain antibody (Ab) regions, and other conjugates into the Env SU of MLV and FeLV has been explored, with relative success (reviewed in reference 16).…”