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The 2012 landmark Science paper demonstrating the use of CRISPR-Cas9 as a gene-editing tool catapulted gene-editing technologies to new levels. Yet, that breakthrough also highlighted precision and efficiency challenges, ushering in an era of improved approaches such as targeted base editing.
In general, base editing induces genetic modifications without relying on double-strand breaks (DSB), resulting in healthier cells that are not forced to undergo a stressful DNA repair response. Less chance also exists for DNA and chromosomal aberrations. Base-editing technologies provide greater precision and traceability than CRISPR-Cas9 and produce a homogenous genotype, within and across the cells.
Modular gene editing
The novel Revvity Pin-point™ base-editing platform is a modular gene-editing, RNA aptamer-mediated technology. The three-part system assembles at the DNA target site through interaction with the guide RNA scaffold.
Advantages of this customizable proprietary base-editing approach include the ability to swap out nucleases and deaminases to increase specificity or to target different genomic regions to use nucleases that therapy developers already have the rights to.
Importantly, the adaptability and modularity support the introduction of multiple discrete modifications, including multiplex knockout and knock-in, simultaneously by utilizing one individual targeting element. This capability facilitates the generation of complex ex vivo gene edited cell therapies as recently demonstrated in a Molecular Therapy publication.1
The paper describes a test case of ex vivo gene editing via the use of a safe, efficient, and fully synthetic Pin-point base-editing system for knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout within a single intervention and without the requirement for additional sequence-targeting components. Furthermore, the aptamer-dependent design overcame the requirement for the delivery of multiple large Cas enzymes by independently controlling which active modules were recruited at each of multiple target loci.1
Delivering the editing machinery
Adeno-associated virus (AAV) delivery systems have demonstrated significant success in preclinical and clinical applications. Although most of the AAV vectors in therapeutic development originate from naturally occurring serotypes, limitations exist such as pre-existing immunity and inherent tropism for certain tissues, along with large-scale manufacturing and dosing requirements.
AAV engineering can address these challenges, resulting in the development of unique, exclusive capsids with improved properties. Revvity offers a collaborative approach and comprehensive support across the entire AAV development pipeline for de novo development of proprietary, licensable, individually tailored AAVs.
AAV-directed evolution and in vivo screening are also employed to modify the AAV capsid surface to assist enhanced transduction and to select capsids with more specific delivery to target cells, leading towards an improved safety profile and reducing dosing and associated costs of these gene therapy vectors.
“The potential of the technology makes us excited about AAV capsid engineering,” said Janina Haar, PhD, Supervisor R&D at Revvity Gene Delivery. “The first modified AAV capsids are being evaluated in the clinic and I look forward to the impact this shift will have on future AAV research and for patients.”
A typical directed evolution project consists of two main phases: an iterative screening campaign followed by a biodistribution study using a subset of vector candidates. Planning includes consideration of library design, selection of AAV serotype, route of administration, and choice of screening model. Once the project is initiated, two to three selection rounds are typically performed in translational large animal models to identify enriched AAV variants.2
In the second phase, promising candidates are produced with individually barcoded expression cassettes for a biodistribution study. This approach allows a quantitative assessment of candidate vector performance at both the genomic and transcriptional levels, providing detailed insight into on- and off-target activity.2
These advanced techniques help drive the development of tailored vectors with improved tissue specificity, altered immune interactions, and enhanced transduction efficiency.
References
1. Porreca I. et al. An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation. Mol Ther. 2024 Aug 7;32(8):2692-2710. doi: 10.1016/j.ymthe.2024.06.033
2. Revvity White Paper. Advancing gene therapy with custom AAV engineering. 2025.
Disclaimer: The Pin-point™ base editing platform technology is available for clinical or diagnostic study and commercialization under a commercial license from Revvity.
