SENT-seq LNP Barcoding for Single-Cell Delivery Profiling
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Solution Overview
Problem
Current methods for identifying lipid nanoparticles (LNPs) suitable for specific cells in vivo are limited by the inability to measure LNP biodistribution, functional delivery, and cellular response at the single-cell level, which hinders the optimization of LNP delivery profiles.
Innovation Solution
The development of Single-Cell Nanoparticle Targeting-sequencing (SENT-seq) methods, which involve formulating LNPs with DNA barcodes and VHH antibodies, administering them to cells, and using sequencing to determine the delivery profile and cellular state at the single-cell level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If in vitro nanoparticle delivery is used to identify candidate genes, then the process is simpler and more controllable, but it does not always recapitulate in vivo nanoparticle delivery and may miss alternative gene candidates
Solution Approach 1:
The patent introduces an intermediary system - a library of LNPs with diverse lipid compositions - to bridge the gap between in vitro and in vivo delivery. This library acts as a mediator that can be screened in vivo to identify gene candidates that truly reflect physiological delivery conditions, resolving the contradiction between simplicity and accuracy by using a structured intermediate approach rather than direct in vivo experimentation
Solution Approach 2:
The patent applies preliminary action by pre-formulating a comprehensive library of LNPs with different lipid compositions before in vivo screening. This preliminary preparation allows systematic evaluation of multiple delivery variants in a controlled manner, enabling accurate gene candidate identification while maintaining experimental manageability through advance planning
2Measurement precision
If single-cell level measurements are implemented to assess LNP biodistribution and delivery, then measurement precision is improved, but the complexity of the assay system increases significantly
Solution Approach 1:
The patent segments the measurement system into distinct functional components: LNP particles segmented by unique DNA barcodes, cellular components segmented by FACS sorting markers, and detection segments separated by sequencing workflows. This segmentation allows each component to be optimized independently while maintaining single-cell resolution, resolving the contradiction between precision and complexity through modular system design
Solution Approach 2:
The patent uses DNA barcodes as information copies that can be amplified and sequenced without requiring direct observation of the LNP-particle itself. This copying approach enables precise tracking of LNP delivery at single-cell level through molecular replication of identification information, achieving high measurement precision while managing system complexity through information-based rather than direct physical measurement
3Productivity
If high-throughput screening methods are used to evaluate multiple LNP formulations, then productivity is improved, but measurement precision at single-cell level may be compromised
Solution Approach 1:
The patent merges multiple measurement dimensions into a unified single-cell assay: biodistribution (via DNA barcode detection), functional delivery (via mRNA expression assessment), and cellular response (via transcriptomic profiling) are all combined in the same single-cell measurement. This merging enables high-throughput screening that maintains single-cell precision by evaluating multiple parameters simultaneously rather than through sequential bulk measurements
Data Source
AI summary
The disclosure provides in vivo methods of identifying a lipid nanoparticle that is optimized based on cellular state, delivery profile, or both for delivery into a specific single cell. The lipid nanoparticles contain an identifying DNA barcode and a VHH antibody. An agent simultaneously detects the DNA barcode, the VHH antibody, and endogenous mRNA of the cell to identify one or more viable cells having the DNA barcode and the VHH antibody at a single cell level. The cellular state of viable cells comprising the lipid nanoparticles is determined by sequencing and measuring reduced expression of one or more of an inflammatory gene, a toxicity gene, and a cell state gene compared to a cell not administered the lipid nanoparticle. Based on a favorable expression profile resulting in the cellular state, the lipid nanoparticle is selected.