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Development of Replicon Cell Pools Bearing a Flavivirus RNA Replicon as a Source of HIV-1 Gag-Pol for Lentiviral Vector Production
by
, , , and
Aitolkyn Kydyrbayeva
,
Viktoriya Keyer
,
Tolganay Kulatay
,
Gulzat Zauatbayeva
,
Bakytkali Ingirbay
,
Maral Zhumabekova
,
Arman Abeev
,
Gaziza Nigmatulla
and
Alexandr V. Shustov
* National Center for Biotechnology, Korgalzhin hwy 13/5, Astana 010000, Kazakhstan
*
Author to whom correspondence should be addressed.
Biology 2026, 15(11), 848; https://doi.org/10.3390/biology15110848 (registering DOI)
Submission received: 16 March 2026
/
Revised: 22 May 2026
/
Accepted: 26 May 2026
/
Published: 28 May 2026
(This article belongs to the Section Biotechnology)
Simple Summary
Gene and cell therapies are innovative medical approaches in which genes or living cells are used as medicines. In many cases, modified viruses known as viral vectors are used to deliver therapeutic genes. An important class of viral vectors is derived from lentiviruses and is widely used in advanced cancer treatments such as CAR-T therapy. However, large-scale production of lentiviral vectors using conventional methods remains challenging, leading to global supply limitations. To address this limitation, we developed a new method for producing lentiviral vectors using self-replicating RNA molecules derived from yellow fever virus. These RNA molecules encode the viral components required for lentiviral vector assembly. We generated cell populations that stably maintain these RNA molecules—termed replicon cell pools—and demonstrated that they can be expanded while continuously producing the required viral components. Although this is a first-generation system, it produces lentiviral vector yields within the same range as current production methods under the conditions tested. With further optimization, this approach could help address the global shortage of viral vectors and enable more affordable access to therapy.
Abstract
Lentiviral vectors (LVs) are indispensable tools in cell and gene therapy. Rising demand has created a global shortage of LVs, driving the development of novel packaging approaches. We report a novel vector packaging approach using autonomously replicating cytoplasmic RNAs (replicons) to express packaging proteins. Yellow fever virus (YFV) was used as a source of replicons encoding the HIV-1 Gag–Pol polyprotein together with reporter or selectable markers. YFV replicons were able to establish chronic infection in HEK293FT cells. Replicons expressing HIV-1 Gag–Pol containing the wild-type HIV-1 protease caused strong cytotoxicity, which prevented the selection of polyclonal cell pools harboring the replicon. In contrast, a replicon carrying the T26S mutation in the HIV-1 protease gene showed no measurable cytotoxic effects, enabling the generation of stable replicon-containing cell pools. The replicon cell pools were established using antibiotic selection and maintained Gag-Pol expression for at least ten passages under selection pressure. Using these first-generation replicon cell pools as packaging cells, LV production required only transient transfection of a transfer vector, a Tat/Rev plasmid, and an envelope plasmid. Yields reached ~106 TU/mL prior to concentration and ~109 TU from multilayer cell stacks, which fall within the range typically reported for conventional transient transfection systems under similar culture conditions. The resulting vectors efficiently transduced target cells, and no replication-competent lentivirus (RCL) was detected using a two-phase RCL assay with p24 ELISA detection. This demonstrator platform utilizing replicon cell pools represents a novel approach for LV packaging.
