CRISPR ACTIVATION GENETIC SCREENING AS A HIGH-THROUGHPUT TECHNOLOGY FOR IMPROVING rAAV PRODUCTION

Recombinant adeno-associated viral (rAAV) vectors are one of the most clinically advanced platforms for gene therapy due to their favorable safety profile, low immunogenicity, and broad tissue tropism. Currently, almost all commercially approved rAAV-based products are manufactured using the triple-transfection protocol in HEK293-derived cells. However, the scalability and productivity of existing manufacturing platforms may not meet the increasing demand for rAAV vectors, highlighting the need for improved production systems. In this context, our laboratory has previously performed a genome-wide CRISPR activation (CRISPRa) screening in ReiCells, a packaging cell line developed by ReiThera S.r.l. and routinely used for clinical-grade adenoviral vector production, to identify cellular genes whose activation enhances rAAV production. This approach identified three gRNAs targeting the promoter of NDUFS2, NFXL1, and TMC1 genes, according to the genomic annotation provided by the human SAM target sequence data (https://www.addgene.org/pooled-library/zhang-human-sam-v2/). These three gRNAs, when simultaneously expressed in SAM-ReiCells carrying the SAM activation system (eSAM-ReiCells), led to an improvement of rAAV vector yield and infectivity up to 6.5-fold across multiple serotypes, highlighting the impact of these gRNAs on rAAV manufacturing. This Master’s thesis describes the generation and characterization of triple-multiplexed eSAM-ReiCells (eSAM-ReiCells 2.0), designed to evaluate whether CRISPR activation using three gRNAs, targeting the same promoter, improves rAAV production compared to the single-gRNA approach previously established. Since eSAM-ReiCells 2.0 were generated through multiple rounds of serial transduction with lentiviral vectors expressing nine gRNAs, targeting the three promoters of the annotated genes (NDUFS2, NFXL1, and TMC1), extensive genomic analyses were performed to verify the stable integration of all gRNAs. To assess the manufacturing capacity of eSAM-ReiCells 2.0, rAAV-DJ was selected for its previously demonstrated high transduction efficiency and productivity. The rAAV-DJ production was compared among HEK293T cells, gold-standard platform for rAAV production, native ReiCells, eSAM-ReiCells and eSAM-ReiCells 2.0. Regardless of whether one or three gRNAs targeting each of the three promoters were expressed, viral production significantly increased compared with native ReiCells, reaching levels comparable to those obtained with the HEK293T. Since the gRNA overload did not further increase viral production, we speculated that the gRNAs identified by the original genome-wide screening may already provide an improvement that reached a plateau under these experimental conditions applied to eSAM-ReiCells 2.0 bulk population. To further investigate possible rAAV manufacturing improvement due to the multiplexed action of different gRNAs, a bunch of single-cell clones were isolated from eSAM-ReiCells and eSAM-ReiCells 2.0. Despite some difference between clones were observed, none overcome the plateau previously identified in eSAM-ReiCells bulk population. It would likely be necessary to isolate a larger number of clones to find one that can improve rAAV production above the bulk average. In conclusion, eSAM-ReiCells and eSAM-ReiCells 2.0 provided rAAV-DJ production levels comparable to those of HEK293T cells, supporting the development of next-generation packaging cell lines with improved productivity, infectivity and quality of rAAV.