Molina Gil, Alberto; (2017) Lentiviral vector packaging cell line development using genome editing to target optimal loci discovered by high throughput DNA barcoding. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Lentiviral vectors are increasingly used as delivery methods in gene therapy clinical trials due to their high efficiency transducing cells and stability of transgene expression. The development of packaging and producer cell lines for the production of lentiviral vectors has always been a labour-intensive and lengthy process. Sequential introduction of vector components, adaptability to suspension cultures, autotransduction and genetic, transcriptional or cell line growth instability are some of the limitations that cause significant drops in productivity. Improved transcription of self-inactivating vectors leading to high titers has been attempted in different ways with the intent to find a high stable producer clone. In this project, we studied the use of lentiviral vectors as a tool to target and identify high-transcribing loci in the genome of our host cells for lentiviral packaging cell line development. Third generation lentiviral vectors carrying eGFP under the control of an endogenous clinically-tested promoter (short EF1α) were produced, containing a variable DNA sequence tag (barcode) in their long terminal repeat (LTR). The aim of the barcode is to uniquely tag, identify and track a particular clone within the heterologous expressing population. Human embryonic kidney cell lines (HEK-293) were transduced with a barcoded lentiviral library at a low multiplicity of infection. We demonstrated that integration site analysis and next-generation sequencing of lentiviral barcoded vector junctions by ligation-mediated PCR (LM-PCR) coupled with RNA-Seq allows for quantification of the relative abundance of each barcode variant in each specific genomic position. Expression cassettes containing lentiviral vector components were then site-specifically integrated into these genomes sites using the CRISPR-Cas9 technology. The barcoding lentiviral system allows for rapid and high-resolution high-throughput screening of gene expression in a large number of genomic positions naturally targeted for optimal vector expression but also of lower expressing sites in order to meet lentiviral cytotoxicity and stoichiometric constraints.

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