C1 - Base Editing and Prime Editing
380: Addressing the dNTP Bottleneck Restricting Prime Editing Activity
Type: Oral Abstract Session
Presentation Details
Session Title: Base Editing and Prime Editing II
The goal of this project is to increase the efficiency of prime editing in post-mitotic cells and tissues. Prime editing has the potential for broad therapeutic application in the treatment of a variety of human diseases due to its versatility in performing targeted base conversions, deletions and insertions. Since the initial description of prime editing, many system components have been further optimized including: Cas9 nickase, M-MLV reverse transcriptase (MMLV-RT), nuclear localization sequences, pegRNA designs and DNA repair pathway activity. The original PE2 system utilizes a pentamutant M-MLV reverse transcriptase (MMLV-RT) that increases thermostability, affinity for the RNA-DNA template and processivity. To improve prime editing outcomes researchers have introduced additional point mutations into MMLV-RT to improve its functionality, substituted alternate RTs in the context of fused or split prime editor systems, or utilized molecular evolution to enhance the properties of multiple RTs for prime editing. Despite these improvements, precise editing rates remain modest in some primary cell types or in some tissue types in vivo. We believe that prime editing efficiency in challenging cellular scenarios can be enhanced by harnessing the wealth of optimization performed on MMLV-RT for in vitro applications. For example, protein aggregation observed during purification of the PEmax prime editor protein appears to be associated with the MMLV-RT domain, which suggests that its solubility may be one limiting factor. Additionally, most characterization and optimization of prime editing systems has been carried out in rapidly dividing mammalian cells, where high intracellular dNTP levels facilitate prime editing efficiency. Cellular dNTP levels can be more than 100-fold lower in post-mitotic or quiescent cells than cycling cells, which would restrict MMLV-RT activity due to its modest affinity for dNTPs. Consequently, we assessed the impact of two MMLV-RT mutations on prime editing activity: V223M, which increases dNTP affinity, and L435K, which increases MMLV-RT solubility. The presence of the L435K mutation translated into improved prime editing for every tested delivery modality (protein, mRNA and plasmid), as well as increasing prime editor protein yields when purifying from bacterial expression systems. Interestingly, the presence of the V223M mutation provided no appreciable benefit to precise editing rates in rapidly dividing transformed cell lines. However, the effect of the V223M mutation was pronounced (~2-fold improvement) in a variety of primary cell types (fibroblasts, induced myoblasts and primary human T cells). Combining the L435K and V223M mutations on the PEmax prime editing framework (PEmax**) yielded 2- to 3-fold improvement in precise editing rates in primary cells and in vivo. To further facilitate MMLV-RT processivity, we knocked down SAMHD1, which is involved in maintaining low intracellular levels of dNTPs, using VPX, a viral accessory protein that targets SAMHD1 for proteasome-mediated degradation. Using VPX with PEmax** increased prime editing rates up to 4-fold over PEmax alone in patient-derived fibroblasts and primary human T cells. We believe that the PEmax** variant in conjunction with SAMHD1 knockdown via VPX co-delivery will have broad utility for precise editing in quiescent and post-mitotic cells.
Pengpeng Liu1, Karthikeyan Ponnienselvan1, Thomas Nyalile2, Sarah Oikemus1, Anya T. Joynt1, Karen Kelly3, Dongsheng Guo4, Zexiang Chen3, Jeong Min Lee5, Celia A. Schiffer5, Charles P. Emerson4, Nathan D. Lawson1, Jonathan K. Watts3, Erik J. Sontheimer3, Luban Jeremy2, Scot A. Wolfe1
1Molecular, Cell and Cancer Biology, UMass Chan Medical School, Worcester, MA,2Molecular Medicine, UMass Chan Medical School, Worcester, MA,3RNA Therapeutics Institute, UMass Chan Medical School, Worcester, MA,4Wellstone Program, Department of Neurology, UMass Chan Medical School, Worcester, MA,5Biochemistry and Molecular Biotechnology, UMass Chan Medical School, Worcester, MA"
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