“Immune Profiling Using Small Volume Blood Samples” in Patent Application Approval Process (USPTO 20240011075): Patent Application

2024 JAN 30 (NewsRx) -- By a News Reporter-Staff News Editor at Insurance Daily News -- A patent application by the inventors Brown, David (Pasadena, CA, US); Dobreva, Tatyana (Pasadena, CA, US); Park, Jong Hwee (Pasadena, CA, US); Thomson, Matthew W. (Pasadena, CA, US), filed on July 11, 2023, was made available online on January 11, 2024, according to news reporting originating from Washington, D.C., by NewsRx correspondents.

This patent application has not been assigned to a company or institution.

The following quote was obtained by the news editors from the background information supplied by the inventors: “

“Field

“This disclosure relates generally to the field of molecular biology, and more particularly to the use of small volume of blood samples for immune profiling.

“Background

“Increasing evidence implicates the immune system in an overwhelming number of diseases, and distinct cell types play specific roles in their pathogenesis. Studies of peripheral blood have uncovered a wealth of associations between gene expression, environmental factors, disease risk, and therapeutic efficacy. For example, in rheumatoid arthritis, multiple mechanistic paths have been found that lead to disease, and gene expression of specific immune cell types can be used as a predictor of therapeutic non-response. Furthermore, vaccines, drugs, and chemotherapy have been shown to yield different efficacy based on time of administration, and such findings have been linked to the time-dependence of gene expression in downstream pathways. However, human immune studies of gene expression between individuals and across time remain limited to a few cell types or time points per subject, constraining our understanding of how networks of heterogeneous cells making up each individual’s immune system respond to adverse events and change over time. There is a need for cost effective, easy-to-access, and non-invasive methods for immune profiling.”

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors’ summary information for this patent application: “Disclosed herein include embodiments of a method for single cell ribonucleic acid sequencing. In some embodiments, the method comprises providing a first low volume, capillary blood sample (or any low volume blood sample and/or any blood sample not obtained from a vein or by venipuncture) obtained from a subject at a first time point. The method can comprise diluting the first sample to obtain a first diluted sample. The method can comprise isolating first capillary peripheral blood mononuclear cells (cPBMCs) from the first diluted sample with gradient centrifugation. The method can comprise performing single cell ribonucleic acid sequencing (scRNA-seq) on the first cPBMCs isolated to generate first scRNA-seq data. The method can comprise determining a first scRNA profile of the subject at the first time point using the first scRNA-seq data and single-nucleotide polymorphisms (SNPs) of the subject.

“In some embodiments, the method comprises providing a second low volume, capillary blood sample obtained from a subject at a second time point. The method can comprise diluting the second sample to obtain a second diluted sample. The method can comprise isolating second cPBMCs from the second diluted sample with gradient centrifugation. The method can comprise performing scRNA-seq on the second cPBMCs isolated to generate second scRNA-seq data. The method can comprise determining a second scRNA profile of the subject at the second time point using the second scRNA-seq data and SNPs of the subject.

“In some embodiments, the first time point and the second time point are about 2 hours to about 24 hours apart. In some embodiments, the subject is in a first health state at the first time point, and the subject is in a second health state at the second time point. The first health state at the first time point can comprise a first disease state of a disease, and the second health state at the second time point can comprise a second disease state of the disease. The first health state at the first time point can comprise first symptoms, and the second health state at the second time point can comprise second symptoms. The first symptoms and the second symptoms can be identical, the first symptoms and the second symptoms can be different, the first symptoms can comprise the second symptoms, and/or the second symptoms can comprise the first symptoms. The first symptoms and the second symptoms can comprise an identical symptom of different severities. In some embodiments, the method comprises receiving the first health state of the subject at the first time point and the second health state of the subject at the second time point. In some embodiments, the method comprises correlating the first health state of the subject at the first time point with the first scRNA profile of the subject at the first time point. The method can comprise correlating the second health state of the subject at the second time point with the second scRNA profile of the subject at the second time point.

“In some embodiments, the method comprises determining a difference between the scRNA profile of the subject at the first time point and the second scRNA profile of the subject at the second time point. The method can thereby determine one or more genes of interest. The one or more genes of interest can comprise diurnal genes. The one or more genes of interest can comprise one or more genes each with a time of day variation in the first scRNA profile and the second scRNA profile. The method can comprise designing a gene panel comprising the one or more genes of interest. The method can comprise determining a difference between the first health state of the subject at the first time point and the second health state of the subject at the second time point. In some embodiments, the method comprises correlating (i) the difference between the scRNA profile of the subject at the first time point and the second scRNA profile of the subject at the second time point and (ii) the difference between the first health state of the subject at the first time point and the second health state of the subject at the second time point.

“In some embodiments, said determining comprises: performing sample demultiplexing of the first scRNA data of the subject and/or the second scRNA data of the subject using SNPs of the subject to determine the first scRNA profile of the subject and/or the second scRNA profile of the subject. In some embodiments, performing sample demultiplexing of the first scRNA data of the subject comprises: classifying scRNA-seq reads with an identical cell label in the first scRNA data as reads generated from a cell of a sample obtained from the subject based on (i) SNPs present in one or more of the scRNA-seq reads with the identical cell label and, (ii) optionally, SNPs of the subject. In some embodiments, performing the sample demultiplexing of the first scRNA data of the subject comprises: classifying scRNA-seq reads with an identical cell label in the second scRNA data as reads generated from a cell of a sample obtained from the subject based on SNPs present in one or more of the scRNA-seq reads with the identical cell label and (ii) optionally, SNPs of the subject. The SNPs of the subject can be determined using the first low volume, capillary blood sample of the subject. In some embodiments, the SNPs of the subject are determined by bulk RNA sequencing and/or scRNA sequencing. Said bulk RNA sequencing and/or scRNA sequencing can be performed using a low volume, capillary blood sample of the subject.

“Disclosed herein include embodiments of a method for single cell ribonucleic acid sequencing. In some embodiments, the method comprises: providing a plurality of low volume, capillary blood samples (or any low volume blood samples and/or any blood samples not obtained from veins or by venipuncture) obtained from a subject at a plurality of time points. The method can comprise, for each of the plurality of samples, diluting the sample to obtain a diluted sample. The method can comprise isolating capillary peripheral blood mononuclear cells (cPBMCs) from the diluted sample with gradient centrifugation. The method can comprise performing single cell ribonucleic acid sequencing (scRNA-seq) on the cPBMCs isolated to generate scRNA-seq data. The method can comprise determining a scRNA profile of the subject at the time point the sample is collected from the scRNA-seq data and single-nucleotide polymorphisms (SNPs) of the subject. The method can comprise determining one or more differences between scRNA profiles of the subject at two or more of the plurality of time points. In some embodiments, two of the plurality of time points are 2 hours to about 24 hours apart, thereby determining one or more genes of interest. The one or more genes of interest can comprise diurnal genes. The one or more genes of interest can comprise one or more genes each with a time of day variation in the scRNA profiles. The method can comprise designing a gene panel comprising the one or more genes of interest.

“In some embodiments, the scRNA-seq comprises a whole transcriptome scRNA-seq. The scRNA profile can comprise a whole transcriptome profile. In some embodiments, the scRNA-seq comprises a target scRNA-seq. The scRNA profile can comprise expression information (e.g., expression profiles) of a plurality of at most 1,000 genes.

“Disclosed herein include embodiments of a method for single cell sequencing. In some embodiments, the method comprises providing a plurality of low volume, capillary blood samples (or any low volume blood sample and/or any blood sample not obtained from a vein or by venipuncture) obtained from a plurality of subjects. The method can comprise isolating immune cells from each of the plurality of samples to obtain isolated immune cells. The method can comprise pooling the isolated immune cells of the plurality of subjects to obtain pooled immune cells of the plurality of subjects. The method can comprise performing single cell sequencing on the pooled immune cells of the plurality of subjects to generate single cell sequencing data of the plurality of subjects. The method can comprise determining a single cell profile of each of the plurality of subjects using the single cell sequence data of the plurality of subjects and single-nucleotide polymorphisms (SNPs) of the plurality of subjects.

“In some embodiments, the method comprises diluting the plurality of samples to obtain a plurality of diluted sample. Isolating the immune cells from each of the plurality of samples to obtain isolated immune cells can comprise isolating the immune cells from each of the plurality of diluted samples to obtain isolated immune cells.

“Disclosed herein include embodiments of a method for single cell sequencing. In some embodiments, the method comprises providing a plurality of low volume, capillary blood samples (or any low volume blood samples and/or any blood samples not obtained from veins or by venipuncture) each obtained from a plurality of subjects. The method can comprise pooling the plurality of samples to obtain a pooled sample. The method can comprise isolating immune cells from the pooled sample to obtain isolated immune cells. The method can comprise performing single cell sequencing on the pooled immune cells to generate single cell sequencing data of the plurality of subjects. The method can comprise determining a single cell profile of each of the plurality of subjects using the single cell sequence data of the plurality of subjects and single-nucleotide polymorphisms (SNPs) of the plurality of subjects.”

There is additional summary information. Please visit full patent to read further.”

The claims supplied by the inventors are:

“1.-17. (canceled)

“18. A method for single cell sequencing comprising: providing a plurality of capillary blood samples obtained from a plurality of subjects; isolating immune cells from each of the plurality of samples to obtain isolated immune cells; pooling the isolated immune cells of the plurality of subjects to obtain pooled immune cells of the plurality of subjects; performing single cell sequencing on the pooled immune cells of the plurality of subjects to generate single cell sequencing data of the plurality of subjects; and determining a single cell profile of each of the plurality of subjects using the single cell sequence data of the plurality of subjects and single-nucleotide polymorphisms (SNPs) of the plurality of subjects.

“19. The method of claim 18, comprising: diluting the plurality of samples to obtain a plurality of diluted sample, wherein isolating the immune cells from each of the plurality of samples to obtain isolated immune cells comprises: isolating the immune cells from each of the plurality of diluted samples to obtain isolated immune cells.

“20. (canceled)

“21. (canceled)

“22. The method of claim 18, wherein the plurality of samples is collected from the plurality of subjects within one week of each other.

“23. The method of claim 18, wherein isolating the immune cells comprises isolating the immune cells with gradient centrifugation.

“24. The method claim 18, wherein the immune cells comprise peripheral blood mononuclear cells (PBMCs).

“25. The method of claim 18, wherein the single cell sequencing comprises: ribonucleic acid (RNA) sequencing, deoxyribonucleic acid (DNA) or DNA-based sequencing, multimaps sequencing and/or exosome sequencing, and/or wherein the single cell profile comprises: an RNA expression profile, a protein expression profile, a multiomics profile, a DNA profile, and/or an exome profile.

“26. The method of claim 18, wherein said determining comprises: performing sample demultiplexing of the single cell sequencing data of the plurality of subjects using SNPs of the plurality of subjects to determine the single cell profile of each of the plurality of subjects.

“27. The method of claim 26, wherein performing sample demultiplexing comprises: classifying single cell sequencing reads with an identical cell in the single cell sequencing data as being from a cell of a sample obtained from a subject based on (i) SNPs present in one or more of the single cell sequencing reads and (ii) optionally, SNPs of the one or more subjects of the plurality of subjects, optionally wherein the SNPs of one or each or the one or more subject are determined by bulk sequencing and/or single cell sequencing, and optionally wherein said bulk sequencing and/or single cell sequencing is performed using a low volume, capillary blood sample obtained from the subject.

“28. (canceled)

“29. The method of claim 18, wherein one, one or more, or each of the plurality of samples has a volume of about 20 ml to about 500 ml.

“30. The method of claim 18, wherein each of the plurality of samples is collected by the subject from whom the sample is obtained from.

“31. The method of claim 18, wherein each of the plurality of samples is collected in a non-clinical setting and/or out of clinic.

“32. The method of claim 18, wherein each of the plurality of samples is collected using a device comprising microneedles, a device comprising microfluidic channels, a push-button collection device, or a combination thereof.

“33. The method of claim 18, wherein each of the plurality of samples is collected from a deltoid or a finger of one of the plurality of subjects from which the sample is collected.

“34. The method of claim 19, wherein said diluting comprises a 1:2 to 1:50 dilution.

“35. The method of claim 19, wherein said diluting comprises diluting each of the plurality of samples having a volume of about 100 ml to about 1 ml.

“36. The method of claim 19, wherein said diluting comprises diluting using a dilution reagent.

“37. The method of claim 36, wherein the dilution reagent comprises a buffer and/or a growth medium.

“38. The method of claim 37, wherein a pH of the buffer is about 7.4, wherein the buffer comprises sodium chloride, potassium chloride, disodium phosphate, monopotassium phosphate, or a combination thereof, wherein a concentration of sodium chloride is about 137 mmol/L, a concentration of potassium chloride is about 2.7 mmol/L, a concentration of disodium phosphate is about 10 mmol/L, and/or wherein a concentration of monopotassium phosphate is about 1.8 mmol/L, and/or wherein the buffer comprises phosphate-buffered saline.

“39. (canceled)

“40. The method of claim 18, wherein said isolating comprises isolating the immune cells with gradient centrifugation using a density medium with a density of about 1 g/ml to about 1.5 g/ml, wherein a duration of the density centrifugation is about 10 mins to about 30 mins, and/or wherein a speed of the density centrifugation is about 500 RPM to about 1500 RPM.

“41. The method of claim 18, said isolating comprises: removing a layer after gradient centrifugation comprising cPBMCs or immune cells, optionally wherein a volume of the layer is about 500 ml to about 1500 ml; and/or removing red blood cells from the layer removed, optionally wherein removing the red blood cells from the layer removed comprises: lysing the red blood cells.

“42.-50. (canceled)”

URL and more information on this patent application, see: Brown, David; Dobreva, Tatyana; Park, Jong Hwee; Thomson, Matthew W. Immune Profiling Using Small Volume Blood Samples. U.S. Patent Application Number 20240011075, filed July 11, 2023 and posted January 11, 2024. Patent URL (for desktop use only): https://ppubs.uspto.gov/pubwebapp/external.html?q=(20240011075)&db=US-PGPUB&type=ids

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