Engineered producer cell lines and methods of making and using the same
Inventors
Tiernan, Aubrey R. • Richards, Nicholas
Assignees
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Publication Number
US-12018288-B2
Publication Date
2024-06-25
Expiration Date
Abstract
This application relates to recombinant adeno-associated virus (rAAV) packaging and/or producer cell lines which have been engineered to reduce expression and/or activity of one or more genes and/or proteins to increase rAAV titers. The methods of generating the engineered cell lines have also been described herein.
Core Innovation
The invention relates to recombinant adeno-associated virus (rAAV) packaging and/or producer cell lines comprising a plurality of engineered cells in which expression of one or more specified host genes is reduced relative to corresponding unmodified parental cells. A core focus is reducing expression of Potassium Calcium-Activated Channel Subfamily N Member 2 (KCNN2) gene product expression to increase rAAV titers.
The engineered cells have reduced expression of KCNN2 and optionally additional specified gene products, including ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, and NALCN-AS1, as compared to corresponding unmodified parental cells. The modulation includes nuclease-based approaches such as CRISPR/Cas9 and RNA interference approaches such as siRNA/shRNA/miRNA and antisense oligonucleotides.
The disclosure also describes methods for generating the engineered packaging/producer cell lines, producing rAAV by infecting the producer cells with helper virus, and harvesting rAAV from lysate and/or cell culture supernatant. Transcriptome-wide differential expression under supplemented versus non-supplemented conditions is used to identify relevant genes, with experimental validation that knocking down or knocking out specified targets including KCNN2 yields statistically significant increases in rAAV titers, including multi-gene knockdowns showing larger fold increases.
Claims Coverage
The independent claim requires an rAAV packaging and/or producer cell line with engineered cells having reduced expression of the KCNN2 gene product compared to unmodified parental cells. Dependent claim coverage further adds multiple additional named gene/RNA targets, specifies modification types, constrains the mechanism for KCNN2 reduction using a guide RNA pair with defined sequence and target DNA sequence constraints, and includes a production method that enhances rAAV production compared to a control parental cell line.
Engineered rAAV packaging/producer cells with reduced KCNN2 expression
An rAAV packaging and/or producer cell line comprising a plurality of engineered cells which have reduced expression of a gene product expressed from Potassium Calcium-Activated Channel Subfamily N Member 2 (KCNN2) as compared to corresponding unmodified parental cells.
Reduced expression of additional specified gene products
The rAAV packaging and/or producer cell line further exhibits reduced expression of specified gene products compared to corresponding unmodified parental cells, including Repulsive Guidance Molecule BMP Co-Receptor A (RGMA), ATP Synthase F1 Subunit Epsilon Pseudogene 2 (ATP EP2), Long Intergenic Non-Protein Coding RNA 319 (LINC00319), Cytochrome P450 Family 3 Subfamily A Member 7 (CYP3A7), ATP Binding Cassette Subfamily A Member 10 (ABCA10), Noggin (NOG), SPANX Family Member N3 (SPANXN3), Pepsinogen A5 (PGA5), Myosin VIIA And Rab Interacting Protein (MYRIP), and NALCN Antisense RNA 1 (NALCN-AS1).
Complete gene deletions of at least one selected gene/RNA
The rAAV packaging and/or producer cell line includes a population of engineered cells having complete gene deletions in at least one of the RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, or NALCN-AS1 genes or RNAs.
KCNN2 reduction using a guide RNA pair with defined sequence and target constraints
The rAAV packaging and/or producer cell line uses engineered cells where KCNN2 expression is reduced by a guide RNA (gRNA) pair, wherein each gRNA either containing nucleotide sequences of SEQ ID NOs: 12-15 and/or targeting a specified target DNA sequence from SEQ ID NOs: 16-19.
Producing rAAV with a helper virus enhances production versus control parental cells
A method for producing rAAV by infecting cells of an rAAV producer cell line with a helper virus enhances production of rAAV in comparison to a control parental cell line.
Overall, the claim coverage centers on engineered rAAV packaging/producer cells with reduced KCNN2 expression versus unmodified parental cells, with dependent coverage extending to additional named gene/RNA reductions, complete gene deletions for selected targets, a constrained gRNA-pair mechanism for KCNN2 reduction using defined SEQ ID NO and target DNA sequence constraints, and a producer-cell infection method that enhances rAAV production relative to control parental cells.
Stated Advantages
Increase rAAV titers.
Statistically significant increases in rAAV titers for knocking down or knocking out specified targets including KCNN2.
Enhancement of rAAV production compared to a control parental cell line.
Documented Applications
rAAV production using an rAAV packaging and/or producer cell line engineered to reduce KCNN2 expression (and optionally additional specified gene/RNA expression), followed by infection with helper virus and harvesting rAAV from lysate and/or cell culture supernatant.
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