Researchers Submit Patent Application, “Immune Profiling Using Small Volume Blood Samples”, for Approval (USPTO 20210324447): Patent Application
2021 NOV 05 (NewsRx) -- By a
No assignee for this patent application has been made.
News editors obtained the following quote 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.”
As a supplement to the background information on this patent application, NewsRx correspondents 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. A method for single cell ribonucleic acid sequencing comprising: providing a first low volume, capillary blood sample obtained from a subject at a first time point; diluting the first sample to obtain a first diluted sample; isolating first capillary peripheral blood mononuclear cells (cPBMCs) from the first diluted sample with gradient centrifugation; performing single cell ribonucleic acid sequencing (scRNA-seq) on the first cPBMCs isolated to generate first scRNA-seq data; and 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.
“2. The method of claim 1, comprising: providing a second low volume, capillary blood sample obtained from a subject at a second time point; diluting the second sample to obtain a second diluted sample; isolating second cPBMCs from the second diluted sample with gradient centrifugation; performing scRNA-seq on the second cPBMCs isolated to generate second scRNA-seq data; and determining a second scRNA profile of the subject at the second time point using the second scRNA-seq data and SNPs of the subject.
“3. The method of claim 1, wherein the first time point and the second time point are about 2 hours to about 24 hours apart.
“4. The method of claim 1, wherein the subject is in a first health state at the first time point, and wherein the subject is in a second health state at the second time point.
“5. The method of claim 4, wherein the first health state at the first time point comprises a first disease state of a disease and the second health state at the second time point comprises a second disease state of the disease, wherein the first health state at the first time point comprises first symptoms and the second health state at the second time point comprises second symptoms, wherein the first symptoms and the second symptoms are identical, the first symptoms and the second symptoms are different, the first symptoms comprise the second symptoms, and/or the second symptoms comprise the first symptoms, and/or wherein the first symptoms and the second symptoms comprise an identical symptom of different severities.
“6. (canceled)
“7. The method of claim 3, comprising: 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; and/or 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.
“8. The method of claim 3, comprising: 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, optionally thereby determining one or more genes of interest, optionally wherein the one or more genes of interest comprise diurnal genes and/or one or more genes each with a time of day variation in the first scRNA profile and the second scRNA profile; designing a gene panel comprising the one or more genes of interest; and/or 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.
“9. The method of claim 8, comprising: 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.
“10. The method of claim 2, wherein 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.
“11.-15. (canceled)
“16. The method of claim 1, wherein the scRNA-seq comprises a whole transcriptome scRNA-seq, and wherein the scRNA profile comprises a whole transcriptome profile.
“17. (canceled)
“18. A method for single cell sequencing comprising: providing a plurality of low volume, 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.-22. (canceled)
“23. The method of claim 18, wherein isolating the immune cells comprises isolating the immune cells with gradient centrifugation.
“24. (canceled)
“25. The method of claim 18, wherein the single cell sequencing comprises: ribonucleic acid (RNA) sequencing, deoxyribonucleic acid (DNA) or DNA-based sequencing, multiomics 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.-28. (canceled)
“29. The method of claim 1, wherein the first sample has a volume of about 20 ml to about 500 ml.
“30. The method of claim 1, wherein the first sample is collected by the first subject.
“31. The method of claim 1, wherein the first sample is collected in a non-clinical setting and/or out of clinic.
“32. The method of claim 1, wherein the first sample is collected using a device comprising microneedles, a device comprising microfluidic channels, a push-button collection device, or a combination thereof.
“33. (canceled)
“34. The method of claim 1, wherein said diluting comprises a 1:2 to 1:50 dilution.
“35.-42. (canceled)
“43. A system comprising: non-transitory memory configured to store executable instructions; and a hardware processor in communication with the non-transitory memory, the hardware processor programmed by the executable instructions to perform: receiving a profile comprising a single cell ribonucleic acid (scRNA) profile of each of a plurality of subjects determined using the method of claim 18; matching a first scRNA profile of a first subject of the plurality of subjects determined from a first sample obtained at a first time point and a second scRNA profile of a second subject of the plurality of subjects determined from a second sample obtained at a second time point, wherein the first time point is prior to the second time point, and wherein a first profile of the first subject comprises a first action performed by the first subject and a first associated outcome occurred subsequent to the action being performed; and generating a report or an output comprising the second scRNA profile, the first action performed by the first subject, the first associated outcome, representations of one or more of the preceding, or a combination thereof.
“44.-50. (canceled)”
For additional information on this patent application, see: Brown, David; Dobreva, Tatyana; Park,
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