Page 91 - IMO-2-1

Innovative Medicines & Omics

SHORT COMMUNICATION
A rapid, efficient, and cost-effective method for

titering third-generation lentiviral vectors

Binhai Ren , Najah T. Nassif , and Ann M. Simpson*
School of Life Sciences, University of Technology Sydney, Sydney, New South Wales, Australia

Abstract

Lentiviral vectors are useful vectors for stable transduction and permanent expression
in dividing and non-dividing cells. In particular, third-generation lentiviral vectors have
been engineered to be significantly safer than their second-generation counterparts,
incorporating several safety features not present in earlier versions. For example, the
tat gene, which is essential for the replication of wild-type human immunodeficiency
virus type 1, has been deleted, and vector packaging functions have been distributed
across three separate plasmids, further enhancing safety. In both research and clinical
settings, having a reliable and accurate method for titering lentiviral vectors is critical.
We have developed a method using the Woodchuck Hepatitis Virus Post-transcriptional
Regulatory Element as a template for a real-time quantitative polymerase chain
reaction, coupled with TRIzol lysis buffer for ribonucleic acid isolation. This method
yielded results comparable to those from a commonly used commercial kit, offering
advantages of speed, cost-effectiveness, and accuracy. It presents a viable, economical
alternative for both research and clinical laboratories.
*Corresponding author:
Ann M. Simpson
(Ann.Simpson@uts.edu.au) Keywords: Titer; Third-generation lentiviral vector; Molecular biology; Ribonucleic acid;
Citation: Ren B, Nassif NT, TRIzol
Simpson AM. A rapid, efficient, and
cost-effective method for titering
third-generation lentiviral vectors.
Innov Med Omics. 2025;2(1):85-92.
doi: 10.36922/imo.6552 1. Introduction
Received: November 24, 2024 Lentiviral vectors are derived from ribonucleic acid (RNA) viruses belonging to the
1st revised: December 20, 2024
2nd revised: December 30, 2024 Retroviridae family. Unlike other retroviruses, lentiviruses can transduce both dividing
Accepted: January 2, 2025 and non-dividing cells, making lentiviral vectors derived from them valuable for a wide
Published online: January 20, range of applications, including gene therapy. Second-generation lentiviral vectors utilize
2025
a single packaging plasmid that encodes the pol, gag, rev, and tat genes, whereas other
Copyright: © 2025 Author(s). virulence factors have been removed. Although these vectors are significantly safer
1-3
This is an Open-Access article than the original lentiviral vectors, the possibility of generating recombinant viruses has
distributed under the terms of the
Creative Commons Attribution not been entirely eliminated. In addition, given that they are almost exclusively derived
License, permitting distribution, from human immunodeficiency virus (HIV), safety concerns persist. In contrast, third-
4
and reproduction in any medium, generation lentiviral vectors mitigate this risk using three separate packaging plasmids:
provided the original work is
properly cited. one for the gag and pol genes, and two for the rev and env genes. Further deletions in
the 3’ long terminal repeat of the vector plasmid make the vectors self-inactivating,
Publisher’s Note: AccScience
Publishing remains neutral with providing an additional safety feature. 4,5
regard to jurisdictional claims in
published maps and institutional Over the past four decades, the number of clinical trials using viral vectors for gene
affiliations. therapy has grown significantly. Throughout this time, numerous breakthroughs have

Volume 2 Issue 1 (2025) 85 doi: 10.36922/imo.6552