Enhanced Lentiviral Vector System: A Platform for Safer Dual-Gene Expression and Pseudovirus Production

University of Pittsburgh researchers have developed a novel enhanced HIV-1 lentiviral vector (LVV) system. This enhanced system consists of a transfer vector, together with three helper plasmids expressing Gag-Pol, Tat, and Rev. This four-plasmid system overcomes many of the limitations of existing lentiviral vector systems and could revolutionize the use of LVVs in both research and clinical applications.

A novel enhanced HIV-1 LVV system has been developed. The system, combining a transfer vector and three helper plasmids is designed to overcome limitations of existing LVV systems.

Description

LVVs are currently used to deliver genes into cells for either research (e.g., vaccine or drug screening) or medical purposes (e.g., gene therapy). LVVs do not contain disease-causing genes and can be extremely useful in producing pseudotyped viruses and various forms of gene therapy. Current LVVs have several limitations including issues with inefficient multi-gene transfer and expression, self-replication, and safety concerns. Through the design of this novel enhanced HIV-1 LVV system, multi-gene delivery has been optimized with independent reporter molecule (luciferase (Luc2) and enhanced green fluorescent protein (EGRP) and, together with the use of helper plasmids, controlled self-replication and self-inactivating processes reduce the risk of adverse events.

Applications

• High-throughput vaccine and antibody screening
• Pseudovirus testing for emerging pathogen research
• Gene therapies

Advantages

Current LVV systems are limited in their ability to deliver and express two genes simultaneously as internal ribosomal entry sites (IRES) are required, limiting expression of the second gene. Additionally, standard LVV systems have known safety risks around unintended viral gene recombination and inconsistent vector yields.

This enhanced LVV system overcomes many of these limitations. Firstly, the transfer vector (pLV-CMV>Luc2-CMV>EGFP), contains two independent CMV promoters, removing the need for IRES. Secondly, the inclusion of two independent promotors allows for dual independent reporting using fluorometric and luminometric analysis to investigate gene delivery. Thirdly, this four-vector system reduces the risk of gene recombination compared to existing two- and three-vector systems. Finally, the inclusion of the truncated HIV-1 long terminal repeats on the transfer vector prevents self-replication post integration.

Invention Readiness

Several enhanced LVV systems have been developed using Nipah, SARS-CoV-2 and other pseudoviruses. Using HEK293T cells, these LVV systems have demonstrated superior reporter expression compared to commercially available LVV systems.

IP Status

Patent Pending

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