Chimeric Ecotropic MLV Envelope Proteins that Carry EGF Receptor-Specific Ligands and the Pseudomonas Exotoxin A Translocation Domain to Target Gene Transfer to Human Cancer Cells

Abstract

Redirecting retroviral vector transduction simply by insertion of a ligand into the envelope (Env) protein has met with several obstacles. For example, virions targeted to epidermal growth factor receptor (EGFR), after receptor binding, rapidly traffic to the lysosomes, where they are degraded. Exotoxin A of Pseudomonas aeruginosa has the ability to translocate from endosomes to the cytoplasm by means of a translocation domain (TLD). We generated a series of chimeric Env proteins of Moloney murine leukemia virus containing EGFR ligands, where TLD was inserted into different regions. These chimeric proteins were successfully produced, if the translocation domain was not located at the immediate N-terminus of Env. The ability to transduce murine cells via the ecotropic receptor varied but correlated with the amount of Env proteins incorporated into the virions. Chimeric vector particles could bind to EGFR, demonstrating the functional exposure of the peptide ligand. However, transduction of human cells expressing EGFR but not the ecotropic receptor by virions carrying the chimeric protein was not observed.

Change of Tropism of SL3-2 Murine Leukemia Virus, Using Random Mutational Libraries

“…Attempts to change the tropism of a virus by engineering its RBD, either by insertion of ligands to known cell surface proteins (16,19,20,27,37) or by selection of randomized libraries (11), have had various degrees of success. Common to all of the previously engineered viral envelopes is that none of them was as efficient as the wild-type envelope.…”

The proline-rich region of the ecotropic Moloney murine leukaemia virus envelope protein tolerates the insertion of the green fluorescent protein and allows the generation of replication-competent virus

“…We inserted GFP at the N terminus and into the proline-rich region (PRR) of Env ( Fig. 1A) because these regions have been shown to tolerate large insertions (Cosset et al, 1995;Kayman et al, 1999;Wu et al, 2000;Erlwein et al, 2002). For insertion into the PRR, the enhanced GFP sequences of plasmid pSFG-EGFP (Lindemann et al, 1997) were amplified by PCR using the primers 59EGFP (59-AAAACC ACCCAGTGGGGTGAGCAAGGGCGAGGAGCTG-39) and 39EGFP (59-AAAACGCCGGTGGACTTGTACAGCTCGT CCATGC-39); GFP sequences are indicated in bold.…”

“…For insertion into the PRR, the enhanced GFP sequences of plasmid pSFG-EGFP (Lindemann et al, 1997) were amplified by PCR using the primers 59EGFP (59-AAAACC ACCCAGTGGGGTGAGCAAGGGCGAGGAGCTG-39) and 39EGFP (59-AAAACGCCGGTGGACTTGTACAGCTCGT CCATGC-39); GFP sequences are indicated in bold. The amplicon was then inserted between the PflMI and SgrAI sites of pwt(HX), an expression plasmid for MoMLV Env (Erlwein et al, 2002). In addition, a unique SnaBI site was engineered for future cloning purposes behind the regenerated SgrAI site by inserting the oligonucleotides 59SNA (59-CCGGTGCGTACGTA-39) and 39SNA (59-CCGGTACGTACGCA-39).…”

Construction of a Gammaretrovirus with a Novel Tropism and Wild-Type Replication Kinetics Capable of Using Human APJ as Entry Receptor

“…Ecotropic envelopes have generally been used as experimental systems for retargeting efforts since their limited tropism (capable of infecting only murine cells) makes them adaptable to such experiments. However, most attempts to produce an envelope protein capable of utilizing a heterologous protein as entry receptor either have been unsuccessful or have resulted in tropism change with severely reduced infection efficiencies (5,13,17,21,31). Only in two cases has efficient retargeting been achieved.…”

“…Several attempts to retarget ecotropic MLV through insertion of single-chain antibodies into SU have failed mainly because the resulting envelopes cannot induce the fusion of membranes after binding to the new receptors. In other cases, insertion of peptide ligands into SU has been used to confer new tropism, usually resulting in very inefficient retargeted envelope proteins (5,13,17,21,31). However, two cases of efficient targeting have been reported, both based on ecotropic MLVs.…”