IP Verse

Spatially Distinguished, Multiplex Nucleic Acid Analysis Of Biological Specimens

Patent No. US11739372 (titled "Spatially Distinguished, Multiplex Nucleic Acid Analysis Of Biological Specimens") was filed by 10X Genomics Sweden Ab on Feb 9, 2023.

What is this patent about?

’372 is related to the field of spatial genomics , specifically methods for analyzing the nucleic acid content of biological samples while preserving spatial information. This addresses the limitation of traditional genomic analysis, which often involves homogenizing samples, thus losing the spatial context of individual cells or regions within a tissue. The background highlights the importance of understanding tumor heterogeneity and the need for tools that can distinguish subpopulations of cancer cells.

The underlying idea behind ’372 is to spatially tag nucleic acids from a biological specimen by hybridizing them to probes with unique barcode sequences attached to a solid support. The location of each probe on the support is determined, and when target nucleic acids from the specimen hybridize to these probes, they are effectively labeled with the barcode corresponding to their location. This allows for subsequent analysis of the nucleic acids while retaining information about their original spatial context within the sample.

The claims of ’372 focus on a method comprising: (a) providing a solid support with randomly located nucleic acid probes, each containing a target capture sequence and a unique spatial tag sequence; (b) determining the spatial tag sequences at each location; (c) contacting the biological specimen with the probes; (d) hybridizing the target capture sequences to target nucleic acids from the specimen; and (e) extending the target capture sequences to produce extended probes containing complementary sequences to the target nucleic acids and the spatial tag sequences.

In practice, the invention involves creating a dense array of probes on a surface, such as a modified Illumina flow cell or a bead array. Each probe contains a unique barcode and a sequence designed to capture specific target nucleic acids, such as mRNA (using oligo-dT sequences). After the barcode locations are mapped using sequencing or hybridization, a tissue section is placed on the array. mRNA released from the tissue hybridizes to the probes, and a reverse transcription step extends the probes, incorporating the mRNA sequence alongside the spatial barcode. The tissue is then removed, and the extended probes, now spatially tagged cDNA, can be analyzed.

This approach differentiates itself from prior methods by directly linking spatial information to nucleic acid sequences at a high density. Unlike traditional methods that rely on physical separation or microdissection, ’372 uses a random array of barcoded probes to capture and tag nucleic acids in situ. This allows for a more comprehensive and spatially resolved analysis of complex biological samples, enabling researchers to study gene expression patterns and genomic variations within specific regions of a tissue or within individual cells.

How does this patent fit in bigger picture?

Technical landscape at the time

In the mid-2010s when ’372 was filed, nucleic acid analysis was typically performed using microarrays or next-generation sequencing platforms. At a time when systems commonly relied on homogenates derived from mixtures of many different cells, preserving spatial information during multiplex nucleic acid analyses was non-trivial. Hardware or software constraints made it difficult to distinguish where any particular nucleic acid resided in the biological specimen.

Novelty and Inventive Step

Claims 2-31 were rejected for obviousness-type double patenting over U.S. Patent Nos. 11,390,912, 11,299,774, 11,162,132, and 10,774,374, and pending U.S. Application No. 17/011,923. The claims were considered free of prior art. The prosecution record does NOT describe the technical reasoning or specific claim changes that led to allowance.

Claims

There are 30 claims in total, with claim 1 being the only independent claim. Independent claim 1 is directed to a method for spatially tagging target nucleic acids of a biological specimen using a solid support with randomly located nucleic acid probes. The dependent claims generally elaborate on and refine the method of independent claim 1, providing details regarding amplification, solid support characteristics, sequencing methods, and biological specimen handling.

Key Claim Terms New

Definitions of key terms used in the patent claims.

Term (Source)Support for SpecificationInterpretation
Extended probes
(Claim 1)		“A method of the present disclosure can include a step of extending solid support-attached probes to which target nucleic acids are hybridized. In embodiments where the probes include barcode sequences, the resulting extended probes will include the barcode sequences and sequences from the target nucleic acids (albeit in complementary form). The extended probes are thus spatially tagged versions of the target nucleic acids from the biological specimen.”The product of extending the target capture sequences after hybridization to target nucleic acids. These probes contain sequences complementary to the target nucleic acids and the spatial tag sequences.
Nucleic acid detection reaction
(Claim 1)		“A method of the present disclosure can include a step of performing a nucleic acid detection reaction on a solid support to determine barcode sequences of nucleic acid probes that are located on the solid support. In many embodiments the probes are randomly located on the solid support and the nucleic acid detection reaction provides information to locate each of the different probes. Exemplary nucleic acid detection methods include, but are not limited to nucleic acid sequencing of a probe, hybridization of nucleic acids to a probe, ligation of nucleic acids that are hybridized to a probe, extension of nucleic acids that are hybridized to a probe, extension of a first nucleic acid that is hybridized to a probe followed by ligation of the extended nucleic acid to a second nucleic acid that is hybridized to the probe, or other methods known in the art such as those set forth in U.S. Pat. No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.”A reaction performed to identify the spatial tag sequences of the nucleic acid probes at their respective locations on the solid support.
Randomly located positions
(Claim 1)		“As used herein, the term “random” can be used to refer to the spatial arrangement or composition of locations on a surface. For example, there are at least two types of order for an array described herein, the first relating to the spacing and relative location of features (also called “sites”) and the second relating to identity or predetermined knowledge of the particular species of molecule that is present at a particular feature. Accordingly, features of an array can be randomly spaced such that nearest neighbor features have variable spacing between each other.”Positions on the solid support where nucleic acid probes are attached without a predetermined order or pattern with respect to the spatial tag sequence.
Spatial tag sequence
(Claim 1)		“As used herein, the term “spatial tag” is intended to mean a nucleic acid having a sequence that is indicative of a location. Typically, the nucleic acid is a synthetic molecule having a sequence that is not found in one or more biological specimen that will be used with the nucleic acid. The location indicated by a spatial tag can be a location in or on a biological specimen, in or on a solid support or a combination thereof. A barcode sequence can function as a spatial tag.”A nucleotide sequence within a nucleic acid probe that identifies the probe's location on the solid support. Each probe at a different location has a unique spatial tag sequence.
Target capture sequence
(Claim 1)		“In particular embodiments, the target capture moiety is a target capture sequence. The target capture sequence is generally complementary to a target sequence such that target capture occurs by formation of a probe-target hybrid complex. A target capture sequence can be any of a variety of lengths including, for example, lengths exemplified above in the context of barcode sequences.”A nucleotide sequence within a nucleic acid probe that is designed to hybridize to a target nucleic acid from the biological specimen.

Litigation Cases New

US Latest litigation cases involving this patent.

Case NumberFiling DateTitle
1:25-cv-01287Oct 21, 202510X Genomics, Inc. v. Illumina, Inc.

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US11739372

10X GENOMICS SWEDEN AB
Application Number
US18166853
Filing Date
Feb 9, 2023
Status
Granted
Expiry Date
Apr 10, 2035
External Links
Slate, USPTO, Google Patents