Lentiviral vectors for gene therapy

The use of lentiviruses as vectors for the delivery of genetic cargo has been well established across many disciplines, from cancer to cariology. Their efficiency has made them valuable tools not only for in vitro gene expression assays and cell functionality tests, but also in a clinical setting as instruments to improve disease outcome. This blog will explore how lentiviral vectors can be used across a range of clinically relevant cell-based gene therapies including CAR-T cell therapy and stem cell therapy, whilst highlighting how PEIpro® transfection reagent has become the gold standard for efficient, reproducible and cost-effective lentivirus manufacturing.


What exactly are Lentiviruses?

Firstly, to understand how lentiviruses work it is important to know the fundamentals of viral biology. The basic objective of any virus is to survive. To achieve this, viruses infect host cells by delivering genetic material into them in order to replicate. Being so efficient at genetic delivery into cells is what makes them such great tools for cell and gene therapy.

Lentiviruses are a subgroup of retroviruses meaning they function by delivering RNA along with reverse transcriptase enzymes, thus providing all the essential machinery for replication. Lentivirus have the capability to integrate into the genome of both dividing and non-dividing cells, giving them an advantage over classic retroviral vectors, which require mitosis for genomic integration.

In order to produce lentiviruses for use in research or clinically, a gene delivery approach is required. Typically, a transfection reagent is used to equip a cell with the machinery to produce vast quantities of your virus of interest which can then be harvested for use. The workhorse cell line used for lentiviral vector production is HEK-293. These cells can be grown adherent or in suspension which offer the possibility to either produce at smaller scale in established adherent production platforms (T-flasks, cell stacks, cell factories), or to produce at larger scale in bioreactors that facilitate scalability and automation of the process.


Improving productivity with PEIpro® gold standard for lentiviral transfection

PEIpro® is a highly efficient polymer-based transfection reagent ideal for DNA delivery, with flexible and scalable viral vector production capabilities. When it comes to gene therapy, it is highly important to have a reliable, easy to use method for virus production, and PEIpro® does just that. Years of research has optimised PEI chemistry to achieve high transfection efficiencies thus resulting in high yields of viral vectors in both adherent and suspension culture systems. Beyond PEIpro®, Polyplus-transfection® has utilised its expertise into the development of higher quality grades of this product, up to PEIpro®-GMP compliant with Pharma GMP guidelines (ICH Q7 and EudraLex Vol 4, Part II and Annex I).

Viral manufacturers bring their extensive expertise for large scale manufacturing of lentiviral vectors, and as such have identified critical parameters to maximise production yields, of which the transfection reagent. PEIpro® meets the needs of viral vector manufacturers such as VIVE Biotech which develop and manufacture lentiviral vectors to treat cancers and rare diseases. With PEIpro®, they have an optimised production process that is scalable and meets regulatory compliance both is US and Europe.

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Lentiviral vectors from transfection to clinical use

CAR-T Cell Therapy

Chimeric antigen receptor T-cell therapy or ‘CAR-T Cell therapy’ involve the use of a patient’s own t-cells as a means of cancer immunotherapy. To achieve this, T cells are collected from a patient’s blood, grown outside the body in laboratory conditions, where they are genetically modified to equip them with cancer targeting antigens. Afterward, the cells are reintroduced into the patient’s bloodstream via IV drip. Thus, the use of CAR-T cells is primarily a cell-based therapy but requires genetic intervention strategies to modify the cells to treat cancer. With this in mind, the use of lentivirus vectors for this purpose has been widely used, owing to the ability of lentiviruses to transduce both non-dividing and dividing cells and given their ability to integrate into the genome provide lasting transgene expression. To date, CAR-T cell therapy which utilised lentivirus transduction has shown remarkable results against many cancer types such as multiple myeloma(1), B Cell leukaemia’s(2) and lymphomas(3), with several FDA approved therapies including axi-cel (Yescarta)(4) and liso-cel (Breyanzi)(5). Evidently, this ever-evolving field of research/therapy shows excellent efficacy and promise. Thus, a reliable transfection reagent such as PEIpro® is critical for robust and scalable production of lentiviral vectors, as illustrated in a proof-of-concept study for production of CD19-CAR lentivirus in small and benchtop scale stirred bioreactors. Read more here: Suspension-based production of lentiviral vectors for CAR T cell therapies.

Hematopoietic Stem cells

Beyond T cell therapy, lentivirus vectors have also shown real promise in the development of effective stem cell therapy platforms. Hematopoietic stem cells (HSC) can be classed as an immature cell with the capacity to differentiate into all types of blood cell, this includes white blood cells (immune cells), red blood cells (oxygen carriers), and platelets (essential for blood clotting). These cells can be harvested and genetically modified in the same way as CAR-T cells, and with this in mind have potential to treat a variety of blood/immune associated diseases.

Lentivirus have been employed successfully for the medication of HSC, having capabilities to restore immune function or correct haemoglobin defects in patients. Diseases in which lentiviral transduced HSC have been used to treat include anaemia(6), Wiskott-Aldrich syndrome(7), and Metachromatic leukodystrophy(8).

To conclude, the use of lentivirus vectors is a powerful tool in cell-based gene therapy to treat a plethora of different diseases. For these therapies to gain momentum, advance research, and to meet regulatory compliance for clinical use, a robust and efficient production process is key, and choosing PEIpro® does just that.



1. Mikkilineni L, Kochenderfer JN. Chimeric antigen receptor T-cell therapies for multiple myeloma. Blood [Internet]. 2017;130(24):2594–602.

2. Picanço-Castro V, Moço PD, Mizukami A, Vaz LD, de Souza Fernandes Pereira M, Silentivirusestre RN, et al. Establishment of a simple and efficient platform for car-t cell generation and expansion: from lentiviral production to in vivo studies. Hematol Transfus Cell Ther [Internet]. 2020 Apr;42(2):150–8.

3. Prommersberger S, Hudecek M, Nerreter T. Antibody‐Based CAR T Cells Produced by Lentiviral Transduction. Curr Protoc Immunol [Internet]. 2020 Mar 4;128(1).

4. https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/yescarta-axicabtagene-ciloleucel.

5. https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/breyanzi-lisocabtagene-maraleucel.

6. Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, et al. Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy. Science (80- ) [Internet]. 2013 Aug 23;341(6148):1233158.

7. Ferrua F, Cicalese MP, Galimberti S, Giannelli S, Dionisio F, Barzaghi F, et al. Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study. Lancet Haematol [Internet]. 2019 May;6(5):e239–53.

8. Fumagalli F, Calbi V, Sessa M, Zambon A, Baldoli C, Rancoita PMV, et al. Lentiviral hematopoietic stem and progenitor cell gene therapy (HSPC-GT) for metachromatic leukodystrophy (MLD): Clinical outcomes from 33 patients. Mol Genet Metab [Internet]. 2020 Feb;129(2):S59.

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