Patent Application
20210255198
A paper copy of the Sequence Listing and a computer readable form of the Sequence Listing containing the file named “2018-032-02_ST25.txt”, which is 1,076 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER), are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID Nos:1-4.
The present disclosure relates generally to methods for assessing high stress states, and predict future clinical events due to high stress, such as psychiatric hospitalizations with stress symptoms, using computer assisted methods and blood gene expression biomarker data. Further, the present disclosure relates to methods for matching individuals with high stress, with medications that can treat stress, and methods for monitoring response to treatment. Finally, the disclosure relates to new methods of use for candidate drugs and natural compounds repurposed for the treatment of stress.
Stress is a subjective sensation. Accordingly, stress disorders (such as PSD) are often not properly diagnosed and treated. Stress disorders, such as post-traumatic stress disorder (PTSD), are prevalent, disabling, and underdiagnosed, in both the military and civilian realm. Stress disorders consist of mental and physical over-reaction to environmental cues that are perceived as potentially harmful, engendered by past exposure to traumatic events. The persistence, intensity, discongruence from the environment, or congruence with excessive response, are all hallmarks of clinical illness. Stress disorders affect one's ability to do things and quality of life. Due to stigma and lack of objective tests, they are often underdiagnosed, sub-optimally treated, and can lead to self-medication with alcohol and drugs. They may culminate in some cases with suicide.
There are no current objective tests to diagnose, so clinicians have to rely on the self-report of patients. An objective blood test for stress will facilitate proper diagnosis and treatment, enabling more confident treatment of those in need of it, without the stigma that it is “all in their head” and “weakness”. Psychiatric patients may have an increased vulnerability to stress, regardless of their primary diagnosis, as well as increased reasons for stress disorders, due to their often adverse life trajectory. As such, they may be a particularly suitable population in which to try to identify blood biomarkers for stress that are generalizable and trans-diagnostic.
Given the negative impact of untreated stress on quality (and quantity) of life, the current lack of objective measures to determine appropriateness of treatment, and the mixed results with existing medications, the importance of approaches such as those of the present disclosure cannot be overstated.
The present disclosure is generally related to biomarkers and their use for tracking stress states and/or predicting a subject's risk of high stress states and/or future psychiatric hospitalizations with stress symptoms. In some embodiments, the biomarkers used herein have been found to be more universal in nature, working across psychiatric diagnoses, genders and subtypes, in other embodiments, the present disclosure relates to biomarkers identified using a personalized approach; that is, by psychiatric diagnosis, gender and subtype.
The present disclosure further relates to drugs for mitigating high stress states in subjects. Particular drugs have been found that can mitigate high stress states in subjects universally; that is, drugs that can be used for mitigating high stress states across psychiatric diagnoses, genders and subtypes of high stress states. Some drugs, however, have been found that can be used more effectively for mitigating high stress states dependent on gender, psychiatric diagnoses, subtypes and combinations thereof.
In one specific aspect, the present disclosure relates to a method of mitigating stress in a subject in need thereof, the method comprising administering a therapy to the subject, the therapy being selected from the group consisting of one or more compounds from Tables 6A-6D.
In another aspect, the present disclosure relates to a method for predicting a high stress state in a subject, the method comprising: obtaining an expression level of at least one blood biomarker from Table 2 in a sample obtained from the subject, obtaining a reference expression level of the blood biomarker, and identifying a difference between the expression level of the blood biomarker in the sample obtained from the subject and the reference expression level of the blood biomarker, wherein the difference in the expression level of the blood biomarker in the sample obtained from the subject and the reference expression level of the blood biomarker indicates a risk for a high stress state in the subject.
The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below.
The present disclosure present disclosure relates generally to methods for assessing high stress states, and predict future clinical events due to high stress, such as psychiatric hospitalizations with stress symptoms, using computer assisted methods and blood gene expression biomarker data. Further, the present disclosure relates to methods for matching individuals with high stress, with medications that can treat stress, and methods for monitoring response to treatment. Finally, the invention relates to new methods of use for candidate drugs and natural compounds repurposed for the treatment of stress.
Furthermore, the predictive ability of the biomarkers discovered were examined, in a completely independent cohort, in all the participants in it, as well as divided by subtypes, and personalized by gender and diagnosis.
In additional embodiments, the present disclosure is directed to drugs for mitigating high stress states in subjects. Particular drugs have been found that can mitigate high stress states in subjects universally; that is, drugs that can be used for mitigating high stress states across psychiatric diagnoses and genders. Some drugs, however, have been found that can be used more effectively for mitigating high stress states dependent on gender, psychiatric diagnoses, and combinations thereof. Exemplary therapies include cefotiam, proguanil, hydroxyachillin, Prestwick-682, levopropoxyphene, isoflupredone, ozagrel, streptozocin, cyclopenthiazide, metformin, corticosterone, calcium folinate, diphenhydramine, ambroxol, xanoterol, botulin, isometheptene, primidone, tocainide, diloxanide, alprostadil, amphotericin B, oxolamine, and combinations thereof.
A powerful longitudinal within-subject design was used in individuals with psychiatric disorders to discover blood gene expression changes between self-reported low stress and high stress states. The list of candidate biomarkers were prioritized with a Bayesian-like Convergent Functional Genomics approach, comprehensively integrating previous human and animal model evidence in the field. The top biomarkers from discovery and prioritization were then validated in an independent cohort of psychiatric subjects with high scores on stress rating scales. The present disclosure identified a list of 116 candidate biomarkers that were nominally significant after the validation step. The candidate biomarkers were then analyzed for their abilities to predict high stress state, and future hospitalizations with stress, in another independent cohort of psychiatric subjects. The biomarkers were tested in all subjects in the test cohort, as well as in a more personalized fashion by gender and psychiatric diagnosis, showing increased accuracy with the personalized approach. The biomarkers were assessed for evidence of involvement in other psychiatric and related disorders, and the biological pathways and networks they are involved in were analyzed. The biomarkers were analyzed as targets of existing drugs for use for pharmacogenomic population stratification and measuring of response to treatment, as well as used the biomarker gene expression signature to interrogate the Connectivity Map database from Broad/MIT to identify drugs and natural compounds that can be repurposed for treating stress.
As used herein, “predicting high stress state in a subject” is used herein to indicate in advance that a subject's stress state will become elevated.
As known by those skilled in the art, “stress state” refers to thoughts, feelings, intent, and behaviors about life and environment, health, financial, and social conditions. “High stress state” refers to scoring in the upper tertile of a visual analog scale for perceived life stress (0 to 100). “Low stress state” refers to scoring in the lower tertile of a visual analog scale for perceived life stress (0 to 100). In some embodiments, the reference expression level of a biomarker can be obtained for a subject who has a low stress state at the time the sample is obtained from the subject, but who later exhibits a high stress state.
As used herein, “a reference expression level of a biomarker” refers to the expression level of a biomarker established for a subject with a low stress state, expression level of a biomarker in a normal/healthy subject with a low stress state as determined by one skilled in the art using established methods as described herein, and/or a known expression level of a biomarker obtained from literature. The reference expression level of the biomarker can further refer to the expression level of the biomarker established for a high stress state subject, including a population of high stress state subjects. The reference expression level of the biomarker can also refer to the expression level of the biomarker established for a low stress state subject, including a population of low stress state subjects. The reference expression level of the biomarker can also refer to the expression level of the biomarker established for any combination of subjects such as a subject with a low stress state, expression level of the biomarker in a normal/healthy subject with a low stress state, expression level of the biomarker for a subject who has a low stress state at the time the sample is obtained from the subject, but who later exhibits a high stress state, expression level of the biomarker as established for a high stress state subject, including a population of high stress state subjects, and expression level of the biomarker can also refer to the expression level of the biomarker established for a low stress state subject, including a population of low stress state subjects. The reference expression level of the biomarker can also refer to the expression level of the biomarker obtained from the subject to which the method is applied. As such, the change within a subject from visit to visit can indicate an increased or decreased stress state. For example, a plurality of expression levels of a biomarker can be obtained from a plurality of samples obtained from the same subject and used to identify differences between the plurality of expression levels in each sample. Thus, in some embodiments, two or more samples obtained from the same subject can provide an expression level(s) of a blood biomarker and a reference expression level(s) of the blood biomarker.
As used herein, “expression level of a biomarker” refers to the process by which a gene product is synthesized from a gene encoding the biomarker as known by those skilled in the art. The gene product can be, for example, RNA (ribonucleic acid) and protein. Expression level can be quantitatively measured by methods known by those skilled in the art such as, for example, northern blotting, amplification, polymerase chain reaction, microarray analysis, tag-based technologies (e.g., serial analysis of gene expression and next generation sequencing such as whole transcriptome shotgun sequencing or RNA-Seq). Western blotting, enzyme linked immunosorbent assay (ELISA), and combinations thereof.
As used herein, a “difference” in the expression level of the biomarker refers to an increase or a decrease in the expression of a blood biomarker when analyzed against a reference expression level of the biomarker. In some embodiments, the “difference” refers to an increase or a decrease by about 1.2-fold or greater in the expression level of the biomarker as identified between a sample obtained from the subject and the reference expression level of the biomarker. In one embodiment, the difference in expression level is an increase or decrease by about 1.2 fold. As used herein “a risk for high stress state” can refer to an increased (greater) risk that a subject will reach a high stress state. For example, depending on the biomarker(s) selected, the difference in the expression level of the biomarker(s) can indicate an increased (greater) risk that a subject will reach a high stress state. Conversely, depending on the biomarker(s) selected, the difference in the expression level of the biomarker(s) can indicate a decreased (lower) risk that a subject will reach a high stress state.
In accordance with the present disclosure, biomarkers useful for objectively predicting, mitigating, and/or preventing high stress states in subjects have been discovered. In one aspect, the present disclosure is directed to a universal method for predicting high stress state in a subject; that is, a method for predicting high stress state across all psychiatric diagnoses and for either gender. The method includes obtaining a reference expression level of a blood biomarker and determining an expression level of the blood biomarker in a sample obtained from the subject. A change in the expression level of the blood biomarker in the sample obtained from the subject as compared to the reference expression level indicates a risk to reaching a level of high stress.
In one embodiment, the expression level of the blood biomarker in the sample obtained from the subject is increased as compared to the reference expression level of the biomarker. It has been found that an increase in the expression level of particular blood biomarkers in the sample obtained from the subject as compared to the reference expression level of the biomarker indicates a risk for high stress state. Suitable biomarkers that indicate a risk for high stress state when the expression level increases can be, for example, one or more biomarkers as listed in Table 2 and combinations thereof.
In another embodiment, the expression level of the blood biomarker in the sample obtained from the subject is decreased as compared to the reference expression level of the biomarker. Suitable biomarkers that indicate a risk for high stress state when the expression level decreases as compared to the reference expression level have been found to include, for example, one or more biomarkers as listed in Table 2 and combinations thereof.
Particularly suitable subjects are humans. Suitable subjects can also be experimental animals such as, for example, monkeys and rodents, that display a behavioral phenotype associated with high stress states. In one particular aspect, the subject is a female human. In another particular aspect, the subject is a male human, and in another particular aspect, the subject is a male depressed human.
A particularly suitable sample for which the expression level of a biomarker is determined can be, for example, blood, including whole blood, serum, plasma, leukocytes, and megakaryocytes.
Various functions and advantages of these and other embodiments of the present disclosure will be more fully understood from the examples shown below. The examples are intended to illustrate the benefits of the present disclosure, but do not exemplify the full scope of the disclosure.
In this Example, biomarkers were assessed for tracking stress states, predicting high stress states, and predicting psychiatric hospitalizations with stress symptoms.
Cohorts
Three independent cohorts were used: discovery (major psychiatric disorders with changes in state stress), validation (major psychiatric disorders with clinically severe trait and state stress), and testing (an independent major psychiatric disorders cohort for predicting state stress, and for predicting trait future hospitalization visits with stress as the primary reason) (
Participants were recruited from the patient population at the Indianapolis VA Medical Center. All participants understood and signed informed consent forms detailing the research goals, procedure, caveats and safeguards, per IRB approved protocol. Participants completed diagnostic assessments by an extensive structured clinical interview-Diagnostic Interview for Genetic Studies, and up to six testing visits, 3-6 months apart or whenever a new psychiatric hospitalization occurred. At each testing visit, they received a series of rating scales, including a self-report visual analog scale (1-100) for quantitatively assessing state stress at that particular moment in time (Simplified Stress Scale—SSS), which has 4-items (Life Stress, Financial Stress, Health Stress and Social Stress). A PTSD Checklist-Civilian Version (PCL-C) scale, which measures clinical severity of trait stress symptoms over the month preceding testing, was also administered. Whole blood (10 ml) was collected in two RNA-stabilizing PAXgene tubes, labeled with an anonymized ID number, and stored at −80° in a locked freezer until the time of future processing. Whole-blood RNA was extracted for microarray gene expression studies from the PAXgene tubes, as detailed below.
For this Example, the within-participant discovery cohort, from which the biomarker data were derived, consisted of 36 participants (28 males, 8 females) with multiple testing visits, who each had at least one diametric change in stress state from low stress state (VAS Life Stress score of ≤33/100) to a high stress state (Life Stress score of ≥67). At least one of the other items (Health Stress. Financial Stress or Social Stress) having concording low or high score with the Life Stress ((
The validation cohort, in which the top biomarker findings were validated for being even more strongly changed in expression compared to the discovery cohort, consisted of 35 male and 13 female participants with both high trait stress (PTSD PCL-C scale scores ≥50, indicating clinically severe stress) and high state stress (VAS Life Stress score of ≥67). (Table 1.
The independent test cohort for predicting state high stress consisted of 95 male and 27 female participants with psychiatric disorders, demographically matched with the discovery cohort, with one or multiple testing visits in the lab, with either low stress, intermediate stress, or high stress (
The test cohort for predicting trait future hospitalization visits with stress symptoms, in the first year of follow-up, and all future hospitalization visits with stress symptoms (
Medications. The participants in the discovery cohort were all diagnosed with various psychiatric disorders, and had various medical co-morbidities (Table 2). Their medications were listed in their electronic medical records, and documented at the time of each testing visit. Medications can have a strong influence on gene expression. However, the discovery of differentially expressed genes was based on within-participant analyses, which factor out not only genetic background effects, but also minimizes medication effects, as the participants rarely had major medication changes between visits. Moreover, there was no consistent pattern of any particular type of medication, as the participants were on a wide variety of different medications, psychiatric and non-psychiatric. Some participants may be non-compliant with their treatment and may thus have changes in medications or drug of abuse not reflected in their medical records. That being said, the goal was to find biomarkers that track stress, regardless if the reason for it is endogenous biology or driven by substance abuse or medication non-compliance. In fact, one would expect some of these biomarkers to be targets of medications. Overall, the discovery of biomarkers with this design occurs despite the participants having different genders, diagnoses, being on various different medications, and other lifestyle variables
TL
NS
FKBP5
1.22E−02/4
FK506
Nominal
Binding
Protein 5
DDX6
L: (13/134)
0.64/4.79E−02
M-SZ
C: (4/29)
0.87/9.64E−03
B2M
F-PSYCHOSIS
C: (4/19)
0.93/4.66E−03
LAIR1
1.12E−02/4
Leukocyte
Nominal
M-PSYCHOSIS
Associated
L: (5/33)
L: (2/95)
Immunoglobulin
0.75/3.94E−02
0.85/4.35E−02
Like
Receptor 1
RTN4
C: (32/398)
0.63/9.49E−03
NUB1
2.34E−02/4
Nominal
C: (38/258)
(6.22E−04/4
0.65/1.42E−03
Top
Nominal)
CIRBP
3.66E−02/4
Cold
Nominal
Inducible
RNA
Binding
Protein
F-BP
L: (2/12)
1/1.58E−02
CYP2E1
1.57E−02/4
Nominal
Male
C: (30/352)
0.63/1.09E−02
MAD1LI
1.47E−02/4
MAD1
Nominal
Mitotic
Arrest
Deficient
Like 1
OAS1
2′-5′-
Oligoadenylate
Synthetase 1
OXA1L
6.40E−03/4
OXA1L,
Nominal
Mitochondrial
Inner
Membrane
Protein
CCL4
C-C
F-PTSD
Motif
C: (3/7)
Chemokine
1/1.69E−02
Ligand 4
DTNBP1
Dystrobrevin
Binding
Protein 1
F-SZA
C: (3/13)
0.93/1.40E−02
SPON2
Spondin 2
ANK2
1.09E−02/4
Nominal
LAIR2
Leukocyte
Associated
Immunoglobulin
Like
Receptor 2
SUMO1
Small
Ubiquitin
Like
Modifier 1
MKL2
4.58E−02/4
Nominal
DMGDH
3.36E−02/4
Nominal
M-SZ
L: (1/44)
1.0/4.52E−02
N4BP2L2
4.40E−02/4
Nominal
M-BP
L: (10/77)
0.74/7.66E−03
PCDHB6
1.17E−02/4
Nominal
Male
C: (25/198)
0.65/7.19E−03
SNCA
Synuclein
M-PSYCHOSIS
Alpha
L: (2/24)
0.98/1.41E−02
M-SZ
L: (2/15)
1/1.36E−02
GJB2
2.42E−02/4
Nominal
M-MDD
C: (6/35)
0.82/7.12E−03
HIF1A
1.11E−02/4
Nominal
PSD3
Pleckstrin
Female
And Sec7
C: (2/46)
Domain
0.98/1.18E−02
Containing 3
STX11
2.74E−02/4
Syntax
Nominal
M-MDD
in 11
C: (5/49)
0.95/4.78−04
APOL3
2.96E−02/4
Apolipoprotein
Nominal
L: (14/234)
L3
0.7/5.34E−03
Male
L: (14/206)
0.71/4.53E−03
ELMO2
1.30E−02/4
Engulfment
Nominal
And
Cell
Motility 2
UBE2E2
4.41E−02/4
Ubiquitin
Nominal
Conjugating
Enzyme E2 E2
FKBP5
FK506
Binding
Protein 5
HLA-
1.22E−02/4
DRB1
Nominal
Major
Histocompatibility
Complex,
Class II,
DR Beta 1
LCP2
2.01E−02/4
Lymphocyte
Nominal
Cytosolic
Protein 2
M-SZA
C: (5/87)
0.85/4.09E−03
M-PSYCHOSIS
C: (8/161)
0.78/3.90E−03
LRRC59
3.15E−02/4
Leucine
Nominal
Rich
Repeat
Containing 59
FOXK2
1.52E−02/4
Nominal
Female
L: (5/33)
0.88/3.89E−03
HLA-B
4.85E−02/4
Major
Nominal
Histocompatibility
Complex,
Class I, B
M-MDD
L: (2/27)
1.0/1.03E−02
NKTR
1.24E−02/4
Nominal
PLEKHA5
3.33E−02/4
Gender/Dx
Nominal
M-SZ
C: (3/74)
0.91/8.24E−03
Clorf123
2.92E−02/4
Chromosome 1
Nominal
Open
Reading
Frame 123
UQCC1
3.33E−02/4
Ubiquinol-
Nominal
M-BP
Cytochrome C
C: (10/101)
Reductase
0.72/1.18E−02
Complex
Assembly
Factor 1
PCBP2
F-BP
C: (6/22)
0.89/3.19 − 03
DCTN5
Dynactin
Subunit 5
LOC105378349
Uncharacterized
M-PSYCHOSIS
LOC105378349
C: (5/47)
0.74/4.22E−02
TL
FKBP5
FK506
Binding
Protein 5
DDX6
B2M
LAIR1
Leukocyte
Associated
Immunoglobulin
Like
Receptor 1
RTN4
NUB1
CIRBP
Cold
Inducible
RNA
Binding
Protein
CYP2E1
MAD1LI
All
MAD1
L: (62/288)
Mitotic
1.8/1.32E−03
Arrest
Deficient
Male
Like 1
(59/255)
1.7/2.66E−03
OAS1
2′-5′-
M-PSYCHOSIS
Oligoadenylate
L: (29/121)
Synthetase 1
2.7/1.52E−02
OXA1L
OXA1L,
Mitochondrial
Inner
Membrane
Protein
F: PSYCHOSIS
C: (6/17)
4.2/3.02E−02
CCL4
C-C
Motif
Chemokine
Ligand 4
DTNBP1
Dystrobrevin
Binding
Protein 1
SPON2
Spondin 2
M-BP
L: (24/91)
4.4/9.90E−04
ANK2
M-MDD
L: (4/32)
76.8/8.14E−03
LAIR2
Leukocyte
Associated
Immunoglobulin
Like
Receptor 2
SUMO1
Small
M-SZ
Ubiquitin
C: (13/93)
Like
2.98/2.98 − 02
Modifier 1
MKL2
DMGDH
N4BP2L2
PCDHB6
SNCA
Synuclein
Alpha
GJB2
HIF1A
PSD3
Pleckstrin
Female
And Sec7
C: (7/53)
Domain
2.2/4.42E−02
Containing 3
STX11
Syntax
in 11
APOL3
Apolipoprotein
L3
ELMO2
Engulfment
And
Cell
Motility 2
M-MDD
C: (9/57)
3.86/8.54E−03
UBE2E2
Ubiquitin
M-PSYCHOSIS
Conjugating
C: (52/201)
Enzyme E2 E2
1.4/5.21E−03
M-SZA
C: (39/108)
1.6/2.83E−03
FKBP5
FK506
Binding
Protein 5
HLA-
DRB1
Major
Histocompatibility
Complex,
Class II,
DR Beta 1
M-SZA
C: (39/108)
1.4/2.18E−02
M-SZA
L: (21/65)
1.7/4.72E−02
LCP2
Lymphocyte
Cytosolic
Protein 2
LRRC59
Leucine
Rich
Repeat
Containing 59
F-SZA
C: (3/12)
56.1/4.25E−02
FOXK2
HLA-B
Major
Histocompatibility
Complex,
Class I, B
NKTR
C: (113/474)
1.4/9.52E−05**
C: (106/421)
1.4/1.43E−04**
M-BP
C: (41/140)
1.6/5.56E−05**
PLEKHA5
Clorf123
Chromosome 1
Open
Reading
Frame 123
Female
L: (3/33)
12.3/3.35E−02
UQCC1
Ubiquinol-
Cytochrome C
Reductase
Complex
Assembly
Factor 1
PCBP2
DCTN5
Dynactin
Subunit 5
M-SZ
L: (8/56)
6.5/4.80E−3
LOC105378349
Uncharacterized
LOC105378349
Bold - indicates biomarker decreased in expression, Italic - indicates biomarker increased in expression. DE-differential expression, AP-Absent/Present. NS- Non-stepwise in validation. Bold name genes also nominally significant at Step 3 validation (n = 29). For Step 4 Predictions, C-cross-sectional (using levels from one visit), L-longitudinal (using levels and slopes from multiple visits). In All, by Gender, and personalized by Gender and Diagnosis (Gender/Dx) M-males, F-Females, MDD-depression, BP-bipolar, SZ-schizophrenia, SZA-schizoaffective, PSYCHOSIS-schizophrenia and schizoaffective combined, PTSD-post-traumatic stress disorder.
RNA extraction. Whole blood (2.5 ml) was collected into each PaxGene tube by routine venipuncture. PaxGene tubes contain proprietary reagents for the stabilization of RNA. RNA was extracted and processed as described in Le-Niculeswu H. et al. Discovery and validation of blood biomarkers for suicidality. Mol Psychiatry 2013; 18(12): 1249-1264; Niculescu A B. et al. Understanding and predicting suicidality using a combined genomic and clinical risk assessment approach. Mol Psychiatry 2015; 20(11): 1266-1285; and Levey D F. et al. Towards understanding and predicting suicidality in women: biomarkers and clinical risk assessment. Molecular psychiatry 2016; 21(6): 768-785.
Microarrays. Microarray work was carried out using previously described methodology (see, Le-Niculescu H. et al., Mol Psychiatry 2013; 18(12): 1249-1264; Niculescu A B, et al., Mol Psychiatry 2015; 20(11): 1266-1285; Levey D F, et al. Molecular psychiatry 2016; 21(6): 768-785; and Niculescu A B et al. Precision medicine for suicidality: from universality to subtypes and personalization. Mol Psychiatry 2017:22(9): 1250-1273).
Telomere Length
Blood was collected in EDTA blood tubes and kept at −80° C. until time of extraction. DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and DNA concentration was assessed using Qubit (ThermoFisher Scientific) as per the manufacturer's protocols. Telomere length (TL) was determined using a relative quantitative real-time PCR (qRT-PCR) method (Mamdani et al. Variable telomere length across post-mortem human brain regions and specific reduction in the hippocampus of major depressive disorder. Transl Psychiatry 2015: 5: e636). Two assays were carried out, one for the Human albumin gene (ALB), which is a single copy gene, and the other assay with primers specific to the repetitive telomeric (TEL) sequence. The primers used to amplify the single copy gene are: ALBF (CTO TCA TCT CTT GTG GGC TOT) (SEQ ID NO:1) and ALBR (GGC ATG ACA GO TIT GCA ATA) (SEQ ID NO:2) and those for the telomeric sequence are: TEL1b (CGG TTT OTT TGG GTT TGG GTT TGG GTT TGG GT TGG GTT) (SEQ ID NO:3) and TEL2b (GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT) (SEQ ID NO:4). A ratio of the relative quantities (TEL/ALB) was used as a quantitative measure of TL Each sample was run in triplicate and an average of the cycle thresholds was used to calculate telomere/single copy gene (T/S) ratios.
Biomarkers
Step 1: Discovery
The participant's score from a visual-analog scale Life Stress, assessed at the time of blood collection (
The data was analyzed in two ways: an Absent-Present (AP) approach, and a differential expression (DE) approach. The AP approach may capture turning on and off of genes, and the DE approach may capture gradual changes in expression. Analyses were performed as described in Niculescu A B, et al., Mol Psychiatry 2015; 20(11): 1266-1285; Levey D F, et al., Molecular psychiatry 2016; 21(6): 768-785; and Niculescu A B et al. Mol Psychiatry 2017; 22(9): 1250-1273.
Gene Symbol for the probesets were identified using NetAffyx (Affymetrix) for Affymetrix HG-U133 Plus 2.0 GeneChips, followed by GeneCards to confirm the primary gene symbol. In addition, for those probesets that were not assigned a gene symbol by NetAffyx, was used GeneAnnot (https://genecards.weizmann.ac.il/geneannot/index.slml) to obtain gene symbol for these uncharacterized probesets, followed by GeneCard. Genes were then scored using a manually curated CPO databases as described below (
Step 2: Prioritization Using Convergent Functional Genomics (CFG)
Databases. Manually curated databases were established of the human gene expression/protein expression studies (postmortem brain, peripheral tissue/fluids: CSF, blood and cell cultures), human genetic studies (association, copy number variations and linkage), and animal model gene expression and genetic studies, published to date on psychiatric disorders. Only findings deemed significant in the primary publication, by the study authors, using their particular experimental design and thresholds, are included in the databases. The databases include only primary literature data and do not include review papers or other secondary data integration analyses to avoid redundancy and circularity. These large and constantly updated databases have been used in the CFG cross validation and prioritization platform (
Step 3: Validation Analyses
Which of the top candidate genes (total CFG score of 6 or above), were stepwise changed in expression from the Low Stress and High Stress group to the Validation Clinically Severe Stress group, were examined. A CFG score of 6 or above reflects an empirical cutoff of 33.3% of the maximum possible total CFG score of 18, which permits the inclusion of potentially novel genes with maximal internal score of 6 but no external evidence score. Participants with Low Stress, as well as participants with High Stress from the discovery cohort, who did not have severe clinical stress (PCL-C <50) were used, along with the independent Validation cohort (n=48).
The AP derived and DE derived lists of genes were combined, and the gene expression data corresponding to them was used for the validation analysis. The cohorts (Validation Clinically Severe Stress, alongside the Low Stress and High Stress groups in the Discovery cohort) were assembled out of Affymetrix.cel data that was RMA normalized by gender and diagnosis. The log transformed expression data was transferred to an Excel sheet, and non-log transformed the data by taking 2 to the power of the transformed expression value. The values were then Z-scored by gender and diagnosis. The Excel sheets were imported with the Z-scored by gender and diagnosis expression data into Partek, and statistical analyses were performed using a one-way ANOVA for the stepwise changed probesets, and stringent Bonferroni corrections was also attempted for all the probesets tested (stepwise and non-stepwise) (
Choice of Biomarkers to be Carried Forward
Top biomarkers from each step were then carried into testing. The list of candidate biomarkers included the top biomarkers from discovery step (≥90% of raw scores, n=39), the top biomarkers from the prioritization step (CFG score ≥13, n=21), and the nominally significant biomarkers after the validation step (n=232), for a total of n=285 probesets (n=269 genes). The biomarkers and trait future hospitalizations with stress in the first year of follow-up, and in all future years of follow-up, were predicted from the list in independent cohorts state (High Life Stress VAS ≥67).
Diagnostics
In Step 4, testing, the test cohort for predicting High Stress (state), and the test cohort for predicting future hospitalizations with stress (trait), were assembled out of data that was RMA normalized by gender and diagnosis. The cohort was completely independent from the discovery and validation cohorts, there was no participant overlap with them. Phenomic (clinical) and gene expression markers used for predictions were Z scored by gender and diagnosis, to be able to combine different markers into panels and to avoid potential artefacts due to different ranges of expression in different gender and diagnoses. Markers were combined by simple summation of the increased risk markers minus the decreased risk markers. Predictions were performed using R-studio. For cross-sectional analyses, marker expression levels, z-scored by gender and diagnosis, were used. For longitudinal analyses, four measures were combined: marker expression levels, slope (defined as ratio of levels at current testing visit vs. previous visit, divided by time between visits), maximum levels (at any of the current or past visits), and maximum slope (between any adjacent current or past visits). For decreased markers, the minimum rather than the maximum was used for level calculations. All four measures were Z-scored, then combined in an additive fashion into a single measure. The longitudinal analysis was carried out in a sub-cohort of the testing cohort consisting of participants that had at least two test visits.
Predicting State High Stress. Receiver-operating characteristic (ROC) analyses between marker levels and stress state were performed by assigning participants visits with a Life Stress VAS score of ≥67 into the High Stress category. The pROC package of R (Xavier Robin et al. BMC Bioinformatics 2011) was used (Table 2,
Predicting Trait Future Hospitalization with Stress as a Symptom/Reason for Admission. Analyses were conducted for predicting future psychiatric hospitalizations with stress as a symptom/reason for admission in the first year following each testing visit, in participants that had at least one year of follow-up in the Veteran's Administration (VA) system. ROC analyses between genomic and phenomic markers measures (cross-sectional, longitudinal) at a specific testing visit and future hospitalization were performed as described above, based on assigning if participants had been admitted to the hospital due to stress or not. Additionally, a one tailed t-test with unequal variance was performed between groups of participant visits with and without future hospitalization with stress. Pearson R (one-tail) correlation was performed between hospitalization frequency (number of hospitalizations with stress divided by duration of follow-up) and marker levels. A Cox regression was performed using the time in days from the testing visit date to first hospitalization date in the case of patients who had been hospitalized, or 365 days for those who did not. The hazard ratio was calculated such that a value greater than 1 always indicates increased risk for hospitalization, regardless if the biomarker is increased or decreased in expression.
Pearson R and Cox regression analyses were also conducted for all future hospitalizations with stress, including those occurring beyond one year of follow-up, in the years following testing (on average 5.76 years per participant, range 0.07 to 11.27 years; see Supplementary Information 2), as these calculations, unlike the ROC and t-test, account for the actual length of follow-up, which varied from participant to participant. The ROC and t-test might in fact, if used, under-represent the power of the markers to predict, as the more severe psychiatric patients are more likely to move geographically and/or be lost to follow-up. The Cox regression was performed using the time in days from visit date to first hospitalization date in the case of patients who had hospitalizations with stress, or from visit date to last note date in the electronic medical records for those who did not.
Biological Understanding
Pathway Analyses
IPA (Ingenuity Pathway Analysis, version 24390178, Qiagen), David Functional Annotation Bioinformatics Microarray Analysis (National Institute of Allergy and Infectious Diseases) version 6.7 (August 2016), and Kyoto Encyclopedia of Genes and Genomes (KEGG) (through DAVID) were used to analyze the biological roles, including top canonical pathways and diseases (Table 3), of the candidate genes resulting from this work. The pathway analyses were run for the combined 220 unique genes (232 probesets) that were nominally significant after validation. For Network analysis of the 220 unique genes, STRING Interaction Network (https://string-db.org) was performed by in putting the genes into the search window and performed Multiple Proteins Homo sapiens analysis.
Tables 3A & 3B: Biological Pathway Analyses of validated biomarkers (n=232 probesets 220 genes). Table 3A. Pathways. Table 3B. Diseases.
A CRG approach was also used to examine evidence from other psychiatric and related disorders, for the list of top predictive biomarkers after Step 4 testing (n=41) (Table 4).
Tables 4A &4B. Methods for Personalized Assessment of High Stress State and Prediction of Risk for Future Clinical Worsening of Stress, such as Hospitalization Related to Stress.
M—males, F—females, BP—bipolar, MDD—Major Depressive Disorder, PTSD—Post-Traumatic Stress Disorder, PSYCHOSIS—schizophrenia or schizoaffective disorder, SZ—schizophrenia, SZA—schizoaffective disorder. D—Decreased in expression; I—increased in expression in high stress states.
Therapeutics
Pharmacogenomics. Which of the individual top predictive biomarkers (n=41) were known to be modulated by existing drugs was analyzed using the CPG databases, and using Ingenuity Drugs analyses (Table 5).
TL
Telomere
Length
Mianserin
Lithium
Reference
marker
Olanzapine
228
Meditation
229, 230
from
Omega-3
Lithium
literature
fatty
acids
FKBP5
1.22E−02/4
FK506
Nominal
Binding
Psychotherapy
Protein 5
Lithium
FKBP5
FK506
Binding
Psychotherapy
Protein 5
Lithium
RTN4
Valproate
Omega-3
OAS1
2′-5′-
Oligoadenylate
Synthetase 1
Lithium
SNCA
Synuclein
Alpha
DBPKO-
Lithium
Stressed
mice,
Omega-3
fatty
acids
B2M
DBPKO-
Stressed,
mice,
Omega-3
fatty
acids
NUB1
2.34E−02/4
Nominal
Clozapine
GJB2
2.42E−02/4
Nominal
Clozapine
HIF1A
1.11E−02/4
Nominal
LRRC59
3.15E−02/4
Leucine
Nominal
Rich
Valproate
Repeat
Containing 59
PSD3
Pleckstrin
And Sec7
Clozapine
Domain
Containing 3
STX11
2.74E−02/4
Syntaxin 11
Nominal
Anti-
depressants
Lithium
Valproate
ANK2
1.09E−02/4
Nominal
Mianserin
HLA-
1.22E−02/4
DRB1
Nominal
Major
Histocompat-
ibility
Complex,
Class II,
DR Beta 1
LAIR2
Leukocyte
Associated
Anti-
Immunoglobulin
depressants
Like
Receptor 2
New drug discovery/repurposing. Drugs and natural compounds were analyzed to determine an opposite match for the gene expression profiles of panels of the top predictive biomarkers, using the Connectivity Map (portals.broadinstitute.org, Broad Institute, MIT) (Table 6). 140 out of the nominally validated 232 probesets from Step 3 were present in the HOU-133A array used for the Connectivity Map. Out of these, gene expression signatures of the probesets that were predictive in Step 4 (nominally significant) were compiled for all participants, as well as separately for males, for females, and personalized by gender and diagnosis.
Tables 6A-6E. New Methods of Use for Therapeutics. Discovery of new method of use for drugs/repurposing. Connectivity Map (CMAP) analysis. Query for signature is done using exact Affymetrix probesets and direction of change. Drugs that have opposite gene expression profile effects to our high stress biomarkers signatures. A score of −1 indicates the perfect match, i.e. the best potential therapeutic for decreasing stress. NIH LINCS analysis using the L1000CDS2 (LINCS L1000 Characteristic Direction Signature Search Engine) tool. Query for signature is done using gene symbols and direction of change. Shown are compounds mimicking the opposite direction of change in high stress. A higher score indicates a better match.
Drug Repurposing Using Connectivity Map (CMAP from Broad Institute/MIT)
Drug repurposing using L1000 Characteristic Direction Signature Search Engine
Convergent Functional Evidence (CPE)
For the top predictive biomarkers (n=42), all the evidence from discovery (up to 6 points), prioritization (up to 12 points), validation (up to 6 points), testing (state, trait first year Hospitalization with Stress visits, trait all future Hospitalization with Stress visits were tabulated into a convergent functional evidence (CFE) score—up to 8 points each if significantly predicts in all participants, 6 points if predicts by gender, 4 points if predicts in gender/diagnosis), other psychiatric and related disorders (3 points), and drug evidence (3 points). The total score can be up to 54 points: 36 from the data and 18 from literature data. The data weighed twice as much as the literature data. The goal was to highlight, based on the totality of the data and of the evidence in the field to date, biomarkers that have all around evidence: track stress, predict it, are reflective of stress and other pathology, and are potential drug targets. Such biomarkers merit priority evaluation in future clinical trials.
Step 1: Discovery of Biomarkers for Stress
A powerful within-participant longitudinal discovery approach was used to identify genes that: (1) change in expression in blood between low stress states (Life Stress VAS≤33 out of 100) and high stress states (Life Stress VAS ≥67 out of 100), (2) track the stress state across visits in a participant, and (3) track stress state in multiple participants. A longitudinally followed cohort of psychiatric participants was used to show diametric changes in stress states between at least two testing visits (n=36 participants) (
It was also examined in the discovery cohort whether subtypes of stress can be identified based on mental state at the time of high stress visits, using two way hierarchical clustering with anxiety, mood, and psychosis measures. Three potential subtypes of stress were identified: predominantly anxious (possibly reflecting increased reactivity), predominantly psychotic (possibly reflecting dis-connectivity), and non-comorbid with other psychiatric symptoms (possibly reflecting better adaptation) (
Step 2: Prioritization of Biomarkers Based on Prior Evidence in the Field
A Convergent Functional Genomics (CFG) approach was used to prioritize the candidate biomarkers identified in the discovery step (33% cutoff, internal score of ≥1 pt) by using all the published prior independent evidence in the field (
Step 3: Validation of Biomarkers for Severe Stress State and Trait
These prioritized candidate biomarkers (n=3,590) were next analyzed in a demographically matched cohort of psychiatric participants with clinically severe state and trait stress, by assessing which markers were stepwise changed in expression from low stress to high stress to clinically severe state and trait stress (
Step 4: Testing for Diagnostics
The top biomarkers from each of the first three steps were carried over for further testing. The list of candidate biomarkers thus includes the top biomarkers from discovery step (>=90% of scores, n=39), the top biomarkers after the prioritization step (total CF score >=13, n=21), and the nominally significant biomarkers after the validation step (n=232), for a total of n=285 probesets (n=269 genes) (
285 candidate biomarkers were tested to determine if they are able to predict stress severity state, and future psychiatric hospitalizations with stress, in another independent cohort of psychiatric participants. Biomarker levels information were used cross-sectionally, as well as expanded longitudinal information about biomarker levels at multiple visits, as predictors. The biomarkers in all participants in the independent test cohort were tested, as well as in a more personalized fashion by gender and psychiatric diagnosis, showing increased accuracy with the personalized approach, in particular in women (
Across all participants tested, NUB1, the top risk biomarker after validation, was also the best predictor for high stress state (AUC 65%, p=0.0014). NUB1 was an even better predictor of stress state by gender in females (AUC 74%, p=0.004), and by gender and diagnosis in female bipolars (AUC 78%, p=0.02). NUB1 (Negative Regulator Of Ubiquitin Like Proteins 1), which was increased in expression in high stress states in this Example, has previous convergent evidence for increase in expression in stress, in human brain (nucleus accumbens in individuals exposed to social isolation before dying) and blood (individuals exposed to combat traumas), as well as in the brain of mice subjected to chronic variable stress. Such reproducibility across studies, tissues and populations provides strong reasons to consider it as a bona fide marker for psychological stress, and it serves as a reassuring de facto positive control for the design and power of this Example. Interestingly, NUB1 is also increased in expression in previous blood biomarker studies of suicide, in both males and females (Table 4). There was a strong clinical connection between stress and suicide.
APOL3 was the best predictor for trait first year future hospitalizations with stress (AUC 70%, p=0.0053). APOL3 was an even better predictor of first year future hospitalizations in males (AUC 71%, p=0.045), and by gender and diagnosis in male depression (AUC 92%, p=0.026). It also is a good predictor of all future hospitalizations with stress in male depression (OR 9.6, p=0.026). APOL3 (Apolipoprotein 13), decreased in expression in high stress states, has previous convergent evidence for decrease in expression in brain in mice subjected to stress. Interestingly, APOL3 is also decreased in expression in previous blood biomarker studies of suicide, in both males and females (Table 4).
MAD1L1 the best predictor for trait all future hospitalizations with stress (OR 1.80, p=0.0013). MAD1L1 was an even better predictor by gender and diagnosis in male bipolar (OR 2.1, p=0.0097) and male depression (OR 31.4, p=0.0055). MAD1L1 (Mitotic Arrest Deficient Like 1), which is decreased in expression in high stress states, has previous convergent evidence for decrease in expression in blood in chronic stress. Of note, MAD1L1 has strong previous genetic and gene expression data for involvement in autism, as well as in bipolar disorder and schizophrenia. It may mediate the impact of stress on those disorders.
NKTR (OR 1.37, p=0.000095) survived Bonferroni correction for all the 285 biomarkers tested. Importantly. NKTR (Natural Killer Cell Triggering Receptor), increased in expression in blood in high stress states, was also reported increased in expression in blood in studies of social isolation in humans, and in brain in studies of chronic variable stress in mice. NKTR is also increased in expression in previous blood biomarker studies of suicide, in both males and females, as well as increased in expression in postmortem brain studies in depression and in schizophrenia (Table 4), possibly underlying the effect of stress in those disorders.
By gender, in females, FOXK2 was the best predictor for state (AUC 88%, p=0.0039), PSD3 the best predictor for trait first year hospitalizations (AUC 98%, p=0.011) and Clorf123 for trait all future hospitalizations (OR 12.26, p=0.033). In males, PCDHB6 was the best predictor for state (AUC 65%, p=0.0072), APOL3 the best predictor for trait first year hospitalizations (AUC 71%, p=0.0045), and MAD1L1 the best predictor for trait all future hospitalizations (OR 1.7, p=0.0027).
Personalized by gender and diagnosis, in female bipolar CIRBP was a strong predictor for state (AUC 100%, p=0.016), and in female schizoaffective HLA-DRB1 for trait all future hospitalizations (OR 39.23, p=0.041). In male schizophrenia. SNCA was a strong predictor for state (AUC 100%, p=0.014), in male depression STX11 was a strong predictor for trait first year hospitalizations (AUC 100%, p=0.00047), and in male depression ANK2 was a strong predictor for trait all future hospitalizations (OR 76.81, p=0.0081).
TL (Telomere Length), used as a comparator/positive control, was a good predictor for stress state and first year hospitalizations, particularly in males with depression (Table 2).
Across all participants tested, and in males, predictions of future hospitalizations with stress were in general somewhat stronger using phenotypic markers (such as the PTSD PCL-C scale and the VAS Stress scale) than biomarkers, but predictions were stronger using biomarkers than phenotypic markers in females, and personalized by gender and diagnosis. Also, panels of the validated biomarkers did not work as well as individual biomarkers, particularly when the later are tested by gender and diagnosis, consistent with there being heterogeneity in the population and supporting the need for personalization (data not shown).
Step 5: Biological Roles
Fifth, the top predictive biomarkers were assessed for evidence of involvement in other psychiatric and related disorders (Tables 2 and 5). A majority of the biomarkers have some evidence in other psychiatric disorders, consistent with the broad effect of stress on the brain and on mind domains/dimensions, whereas a few seem to be specific for stress, such as HLA-B (Major Histocompatibility Complex, Class 1, B), LOC105378349 (Uncharacterized LOC105378349), and STX11 (Syntaxin 11). More than half of the top predictive biomarkers (26 out of 41 genes, i.e. 63%) have prior evidence for involvement in suicide, suggesting an extensive molecular co-morbidity between stress and suicide, to go along with the clinical and phenomenological co-morbidity.
The biological pathways and networks in which the nominally validated biomarkers (n=232 probesets 220 genes) are involved were further analyzed. The top biological pathway is involved in antigen processing and presentation (Table 3), broadly speaking in the reaction to threats. The pathways are shared with other non-psychiatric diseases, suggesting that stress is a whole-body disease. There is a network centered on HLA DRB1 that may be involved in reactivity/immune response. A second network is centered on HDAC3, and may be involved in activity/trophicity. A third network is centered on RACI, and may be involved in connectivity/signaling. ACTR1A seems to be a nodal gene connecting these three networks. (
Step 6: Targeted Treatments and Drug Repurposing
Sixth, the top predictive biomarkers as modulated by existing drugs (Tables 2 and 6) was analyzed. The validated biomarker signature, and out of them, the top predictive biomarkers gene expression signatures, were used to interrogate the Connectivity Map database from Broad/MIT to identify drugs and natural compounds that have the opposite effects on gene expression to stress, and can be repurposed for treating stress (Table 6). Reversing the gene expression signature in essence increases the expression of the resilience genes and decreases expression of the risk genes. The top drugs and nutraceuticals identified as potential new stress therapeutics are cefotiam (an antibiotic) and calcium folinate (a B vitamin) using all the validated biomarkers, ambroxol (originally a mucolytic drug, with recent evidence sodium channel blocker with anti-pain properties) and betulin (a triterpene compound from the bark of the birch tree, with evidence for anxiolytic effects) in all using the predictive biomarkers, as well as ozagrel (an antiplatelet agent working as a thromboxane A2 synthesis inhibitor) in males and flecainide (an antiarrhythmic agent that blocks sodium channels) in females.
Step 7: Convergent Functional Evidence (CFE)
The biomarkers with the best overall convergent functional evidence (CFE) across the six steps were FKBP5, DDX6, B2M, LAIR1, RTN4 and the previously mentioned NUB1 (Table 1). FKBP5 (FK506 Binding Protein 5), a decreased in expression biomarker, survived discovery, prioritization and validation. It seems to be a better predictor for state in females, and for trait in males, especially personalized by diagnosis. FKBP5 has independently been described as decreased in expression in blood in World Trade Center attack survivors and in a Dutch cohort with post-deployment PTSD30, as well as in postmortem brains from PTSD. FKBP5 appearance in the present screen is reassuring and serves as a de facto positive control for the approach. It is also involved in multiple other psychiatric disorders, consistent with the role of stress as a trigger or precipitant of illness (Table 4). There is previous evidence for its modulation in expression in opposite direction to stress by mood stabilizers (Table 3), and interestingly, by psychotherapy. DDX6 (DEAD-Box Helicase 6), an increased in expression biomarker, has previous convergent evidence of being increased in expression in blood and in amygdala of mice subjected to stress. It is a strong predictor of state and trait stress across all, by gender, and by gender and diagnosis. DDX6 has also been implicated in other neuropsychiatric disorders (alcoholism, other addictions, depression, schizophrenia), as well as is an increased in expression blood biomarker for suicide in previous studies. LAIR1 (Leukocyte Associated Immunoglobulin Like Receptor 1), a decreased in expression biomarker, survived discovery, prioritization and validation. It has previous convergent evidence from human studies of being decreased in expression in blood in PTSD related to childhood trauma and to interpersonal trauma in females. It is a strong predictor of state stress in females, and of trait stress across all and in males. LAIR1 is also a decreased in expression blood biomarker for suicide in previous studies. RTN4 (Reticulon 4), an increased in expression biomarker, has previous convergent evidence of being increased in the nucleus accumbens (NAC) in social isolation in humans, and in blood in PTSD. It is decreased in expression in blood by treatment with the nutraceutical omega-3 fatty acid DHA in stressed female mice in independent studies, as well as by valproate in brain of mice. RTN4 is a predictor of trait future hospitalizations with stress in all, as well as separately in males and females. RTN4 has also been implicated in bipolar disorder, alcoholism, and pain, as well as is an increased in expression suicide blood biomarker in our studies. B2M (Beta-2-Microglobulin), an increased in expression biomarker, has previous convergent evidence of being increased in the nucleus accumbens (NAC) in social isolation in humans, and it is decreased in expression in NAC by treatment with the nutraceutical omega-3 fatty acid DHA in stressed female mice in independent studies. It is a strong predictor of state stress in females with psychotic disorders, and of future hospitalizations with stress in both genders. B2M has also been implicated in other neuropsychiatric disorders (alcoholism, autism, depression, eating disorders, pain, as well as aging and suicide), possibly mediating the effects of stress in those disorders.
This application claims priority to U.S. Provisional Application Ser. No. 62/683,320, filed on Jun. 11, 2018, which is hereby incorporated by reference in its entirety.
This invention was made with government support under OD007363 awarded by the National Institutes of Health and CX000139 merit award by the Veterans Administration. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/035513 | 6/5/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62683320 | Jun 2018 | US |
