Spatially encoded biological assays

Patent No. US10996219 (titled "Spatially encoded biological assays") on Jun 7, 2019. The application was issued on May 4, 2021.

’219 is related to the field of spatially encoded biological assays and high-resolution mapping of biological molecules. The technology addresses the challenge of measuring the abundance and activity of numerous genes or proteins simultaneously while preserving the context of their specific locations within a solid sample, such as a tissue section. Traditional methods like laser capture microdissection or in situ PCR often lack the scalability or spatial resolution required for comprehensive transcriptomic or proteomic mapping.

The underlying idea behind ’219 is the decoupling of biological target detection from spatial localization through a digital encoding scheme. By using probes that consist of a target-specific binding agent—such as an antibody or oligonucleotide—conjugated to a unique nucleic acid tag, the invention allows biological interactions to occur in situ. These tags act as spatial barcodes that can be collected, pooled, and read using high-throughput sequencing, effectively converting spatial information into a digital dataset that can be mapped back to the original sample geometry.

The claims of ’219 focus on a method for determining the presence of a target biological molecule at a specific region of interest within a tissue sample. The process involves delivering a plurality of probes to the sample, where each probe features a capture agent (like an antibody or aptamer) conjugated to an oligonucleotide. After the capture agent binds to its target, the system separates bound probes from unbound ones, removes the oligonucleotide from the region of interest, and sequences it to confirm the target's presence at that specific location.

In practice, the invention utilizes controlled reagent delivery, such as inkjet printing or microfluidics, to apply these encoded probes or subsequent encoding agents in defined spatial patterns. For example, a combinatorial grid can be created by applying one set of tags in horizontal rows and another in vertical columns. When a probe is recovered with a specific combination of tags, its precise coordinate-based origin within the tissue is revealed. This allows for the simultaneous analysis of thousands of targets across thousands of locations without requiring a unique physical well for each reaction.

This approach differs from prior methods by combining the high multiplexing capabilities of next-generation sequencing with the spatial integrity of histology. Unlike microarrays that require physical transference of tissue to a pre-fabricated grid, this system allows for random access to the sample and can follow the natural topology of the tissue. By reducing the volume of each assay to the level of small cell clusters or even single cells, the invention significantly increases the signal-to-noise ratio, enabling the detection of rare somatic mutations that would otherwise be lost in bulk sample processing.

How does this patent fit in bigger picture?

Technical Landscape

In the early 2010s when ’219 was filed, the analysis of gene and protein expression patterns in biological samples was typically implemented using microarrays, qPCR, or in situ hybridization. At a time when these systems commonly relied on bulk processing of homogenized tissue or the physical transfer of micro-dissected samples into discrete reaction wells, the ability to achieve high levels of multiplexing while maintaining spatial resolution was severely limited. Furthermore, when hardware and software constraints made the simultaneous measurement of thousands of targets across a continuous tissue section non-trivial, researchers were often forced to choose between high-throughput digital quantification and the preservation of the original spatial context of the biological molecules.

Prosecution Position

The disclosed invention represents a meaningful technical advancement through the integration of a spatial encoding scheme with high-throughput digital sequencing to create high-resolution maps of biological activity. This architectural shift moves away from physical compartmentalization of samples and instead utilizes encoded probes comprising a target-binding region and a location-specific coding tag. This capability enables the simultaneous detection of a vast number of biological targets—including nucleic acids and proteins—across multiple sites in a sample by pooling the probes for parallel sequencing and subsequently mapping the digital readout back to specific spatial coordinates. This approach overcomes the technical constraint of low spatial resolution in multiplexed assays, allowing for the identification of molecular distributions at the scale of individual cells or small cell groups without the labor-intensive requirements of laser capture microdissection.

Claims

This patent contains a total of 30 claims, with claim 1 serving as the sole independent claim. The independent claim focuses on a method for determining the presence of a target biological molecule within a specific region of a tissue sample by delivering probes with oligonucleotide-conjugated capture agents, separating bound probes from unbound ones, and sequencing the removed oligonucleotides to identify the target. The dependent claims serve to specify various capture agents such as antibodies or nucleic acids, define target molecules like mRNA or proteins, detail the use of high-throughput sequencing and amplification techniques, and describe applications involving multiple regions of interest, histological imaging, and the generation of three-dimensional molecular maps.

Key Claim Terms New

Definitions of key terms used in the patent claims.

Term (Source)	Support for Specification	Interpretation
Capture agent (Claim 1)	The assay systems utilize one or more binding agents that specifically bind to the biological molecule of interest and unique coding identifiers associated with specific binding agents. In some aspects the probe regions of the encoded probes comprise antibodies, aptamers or small molecules. In particular aspects of the invention the biological targets comprise nucleic acids and the encoded probes are oligonucleotides.	A molecule-specific binding agent, such as an antibody, aptamer, or oligonucleotide, designed to interact with and bind to a specific target biological molecule.
Oligonucleotide (Claim 1)	In some aspects, the multiple biological targets comprise proteins, the probe regions of the encoding probes are proteins and the coding tags comprise oligonucleotides. This encoding scheme uses both molecule-specific binding agents and coding identifiers to provide a practical and cost-effective determination of information on multiple biological molecules. The encoding scheme of the systems can be controlled by delivery of different reagents to discrete regions on the substrate surfaces.	A nucleic acid sequence conjugated to a capture agent that serves as a coding tag or identifier to facilitate the digital readout and identification of the target or its location.
Region of interest (Claim 1)	The assay systems of the invention detect the presence or absence and relative amount of a biological target or biological activity indicative of a biological target, as well as the location of the biological target or activity in a biological sample, e.g., a tissue section or other biological structure disposed upon a support. In some aspects, the known spatial pattern is determined by histological features of the sample. The assay results such as the amount or activity of biological targets can then be mapped back to specific location in the biological sample.	A specific spatial location or site within a tissue sample, often defined by histological features or a known spatial pattern, where biological activity is assayed.
Sequence (Claim 1)	“Sequencing”, “sequence determination” and the like means determination of information relating to the nucleotide base sequence of a nucleic acid. In one aspect, the term includes the determination of the identity and ordering of a plurality of contiguous nucleotides in a nucleic acid. Decoding of the encoding scheme can be performed by, e.g., next-generation sequencing, which easily provides millions to trillions of data points at low cost.	The nucleotide base information of an oligonucleotide, determined via sequencing to decode the identity or location of a bound target molecule.
Target biological molecule (Claim 1)	The biological molecules to be detected can be any biological molecules such as proteins, nucleic acids, lipids, carbohydrates, ions, or multicomponent complexes containing any of the above. Further examples of subcellular objects include organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplast, endocytic vesicle, exocytic vesicles, vacuole, lysosome, etc.	A specific molecule of interest within a biological sample, such as a nucleic acid, protein, peptide, carbohydrate, lipid, or enzyme, whose presence, abundance, or activity is being measured.

Litigation Cases New

US Latest litigation cases involving this patent.

Case Number	Filing Date	Title
1:23-cv-01375	Dec 1, 2023	10X Genomics, Inc. V. Curio Bioscience, Inc.

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US10996219

Application Number: US16435295A
Filing Date: Jun 7, 2019
Publication Date: May 4, 2021
External Links: Slate, USPTO , Google Patents

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