Large scale lentiviral vector production

Lead Research Organisation: University College London
Department Name: Haematology

Abstract

Lentiviral vectors (LV) are remarkable in their ability to insert their genetic payload into a target cell's genome. This affects a permanent genetic change in the target which is propagated through to its progeny. While there are several means of genetic modification, few are capable of permanent modification of target cells. Amongst all vectors for gene delivery, LV are unique in unparalleled efficiency, safety, lack of toxicity and ability to modify non-dividing target cells. They have therefore come to be recognised as a key reagent required for the efficient development of the burgeoning cell therapy, as well as gene therapy industries. However, although LV represent a well understood and robust technology, there is no manufacturing methodology for very large-scale LV production. This is now an acknowledged bottle-neck both for clinical trials and for commercial exploitation of many cell and gene therapy products in current development. We propose to address this unmet need

Technical Summary

* Lentiviral vectors (LV) are used to genetically modify target cells. The main commercial application of LVs is in vitro production of cellular therapeutics, although they can be used as therapeutic agents in their own right and for generation of protein producer cells. While small-scale production is well established, a process for very large-scale production has not been developed. Recently, commercial demand for LV has increased greatly. Large-scale production is a major unmet need and its lack a bottleneck esp. in the marketing of cellular therapies. The challenge of large-scale LV production is multifaceted, requiring a deep understanding of lentiviral biology, as well as skilful genetic and cellular engineering, appreciation of regulatory / GMP considerations, industrial/pharmaceutical scale production issues as well as an understanding of the down-stream clinical applications.

Planned Impact

As described in proposal submitted to IUK

Publications

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Mekkaoui L (2018) Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell. in Molecular therapy. Methods & clinical development

 
Description We identified the expression density required for stable lentiviral vector production. We generated a stable CAR T gene-therapy producer from the WinPAC cell line which was a fallback strategy to meet the main objective of the project which had an acceptable titer. Work is ongoing with alternative funding.
Exploitation Route Useful base for stable cell line for lentiviral production
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title Anti-RD114 monoclonal antibody 
Description Rat monoclonal antibody against the RD114 envelope glycoprotein. Until now, only polyclonal goat antibodies were available for this important retroviral envelope. Further, there was not a stable supply. Since this is a limitation in the field of gene-therapy, we generated a monoclonal antibody against RD114 envelope by making a hybridoma. We sequenced the hybridoma and make a recombinant antibody. The heavy and light chain sequences are in the public domain and the plasmids available through addgene.org 
Type Of Material Antibody 
Year Produced 2019 
Provided To Others? Yes  
Impact This facilitates the generation of high-titer retroviral and lentiviral producer clones by screening cells which have high level of expression. It also allows for blocking of infectivity of these particles which may be of use in some veterinary applications. ADDGENE:75178 and ADDGENE:72029 
 
Description Collaboration with KCL for large-scale lentiviral vector production 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided cell line and genome engineering skills.
Collaborator Contribution Partner brought expertise in large-scale vector processing and purification.
Impact Scaled up method for lentiviral purification based on streptag / streptavidin purification.
Start Year 2017
 
Title METHOD AND MEANS FOR PURIFYING RETROVIRAL VECTORS 
Description The present invention relates to a producer cell which expresses a tagging protein at the cell surface, such that retroviral vectors produced by the cell are tagged with the tagging protein, wherein the tagging protein comprises: i) a binding domain which binds to a capture moiety ii) a spacer; and iii) a membrane targeting domain such that, when incorporated a retroviral vector, the tagging protein facilitates purification of the retroviral vector from cellular supernatant via binding of the tagging protein to the capture moiety. The present invention also relates to a retroviral vector comprising such a producer cell-derived tagging protein. 
IP Reference US2017240920 
Protection Patent application published
Year Protection Granted 2017
Licensed No
Impact Useful methodology
 
Title NUCLEIC ACID CONSTRUCTS FOR PRODUCING RETROVIRAL VECTORS 
Description The present invention relates to a nucleic acid construct comprising: (i) a first nucleic acid sequence which either comprises a retroviral transfer vector or which encodes a retroviral protein; and (ii) a second nucleic acid sequence which encodes a detectable marker which is a cell surface protein comprising an extracellular domain and a membrane targeting domain. 
IP Reference US2019055568 
Protection Patent application published
Year Protection Granted 2019
Licensed No
Impact Useful method