Lentiviral vectors are central to the application of many exciting gene therapy strategies, including gene-supplementation (e.g. SCID) and immunotherapies such as CAR T cells, used in cancer treatment.
Two factors currently limiting wider development of these approaches are the yield and scalability of lentiviral production systems, and relatively low infectivity in T cells. We set out to systematically improve these limitations.
Vector design for transient expression
A proprietary vector set was developed around the 3rd generation lentiviral production system, using Oxford Genetics’ SnapFast vector technology. Initially expression cassettes were produced on four independent plasmids, and subsequently combined onto a reduced, 3-plasmid system (see below).
A number of key characteristics were taken into account during design of the system:
- Vector backbone should be minimised and use the KanR selection marker
- DNA should be fully synthetic and traceable
- Codon optimisation should be used to reduce homology with selected viruses (HIV/VSV)
- Expression cassettes should be designed to minimise inter-cassette homology by using distinct promoters/UTRs/introns/poly A sequences
Optimization of vector ratios to maximize production
The four vector system was tested when each component was titrated out, in order to identify the ratio of vectors giving the highest level of infectious lentiviral particle production.
Lentiviral particles generated using an eGFP genome construct were harvested 72hrs after transfection of HEK293T cells, and titrated also using HEK293T.
The percentage GFP+ve cells was analyzed by flow cytometry relative to un-infected cells. This system was benchmarked against a number of commercial alternatives and a showed a significant increase in production levels.
Additional improvements in productivity have been seen by pairing two components on a single vector to create a 3-vector packaging system.
Generation of stable VSV-G suspension cell lines using expression constructs co-expressing ancillary proteins
Oxford Genetics has carried out extensive screening to identify genes (>1000 screened to date) from a diverse range of organisms, that when co-expressed with the GOI, support protein production. Here, a subset of these have be co-expressed with the VSV-G gene, as part of constructs designed for stable cell line generation.
Stable VSV-G expressing cell lines were produced using antibiotic selection in a suspension adapted HEK293 cell line. Recovery from selection was monitored over time, and indicated that a number of the ancillary proteins supported stable pool generation.
Representative data is shown in the adjacent figure. Further optimization of pool generation is currently in progress.
Characterization of stable VSV-G suspension cell lines
The stable VSV-G cell lines were characterized by cell surface staining for VSV-G expression, and by transient expression experiments, both with and without the vector encoded VSV-G. This demonstrated that the integrated copy can substitute for the vector copy, and that good yields were achieved.
The reduced levels without the VSV-G on the vector reflect the high dosage of the VSV-G gene from the transient transfection.
Once all of the component for lentiviral packaging are integrated into the host cell genome, it is anticipated that the levels achieved here will be sufficient for extended duration fed-batch culture production modes.
VSV-G stable cell lines were analyzed by cell surface staining and flow cytometry analysis. High levels of VSV-G expression were observed in a range of the VSV-G cells lines.
Analysis of clonal derivatives for each of the high expressing stable pools is underway, in order to provide information on homogeneity of the pools, isolation of ‘super-expressers’ and to provide lines suitable for stability analysis.
Oxford Genetics has further designed both inducible and constitutive expression constructs to create full packaging and producer cell lines (featuring all required packaging components).
The packaging lines, which currently in development, will form the basis of an internal and licensable cell line development platform (see below).
Cell line development platform
Full cell line development platforms are being established at Oxford Genetics for generation of stable pool and clonal suspension-mode packaging cell lines suitable for cGMP manufacture. Processes have been designed to meet both regulatory requirements and technical specifications for large scale bioproduction.
Production of pseudotyped lentiviral particles
A literature search was performed to identify and obtain sequences for a range of different glycoproteins (circa. 50), from both RNA and DNA viruses. These were substituted for the VSV-G gene in the lentiviral packaging vectors, either individually or in combination based on the predicted requirements for infectivity.
The lentiviral particles were produced by standard methods using adherent 293T cells, and harvested 72hrs following transfection. Information on a subset of these glycoproteins is summarised below.
Analysis of pseudotyped lentiviral particle infectivity in T and B cells
The pseudotyped lentiviral particles were used to infect monocyte depleted PBMCs that were activated for 72hrs with CD3/CD28 beads for analysis of T cell infectivity, and unactivated for analysis of B cell infectivity. The infections were carried out with defined volumes, and thus reflect production and infection characteristics of the particles.
Data indicates the % GFP positive cells in the CD3+ve and CD19+ve populations, and is normalised to data for VSV-G particles. Further analysis is on-going, both with the larger glycoprotein panel and utilising normalisation to physical titre via qPCR.
