pejman naraghi arani, phd - aacc

LLNL‐PRES‐548771This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract DE‐AC52‐07NA27344. Lawrence Livermore National Security, LLC

Presented to AACC Oak Ridge ConferenceApril 20, 2012

OFFICIAL USE ONLY

UNCLASSIFIED

UNCLASSIFIED

Pejman Naraghi‐Arani, [email protected]

Lawrence Livermore National LaboratoryLLNL‐PRES‐548771

2

We are developing a direct hybridization assay thatsimultaneously detects 19 Category A, B, and C virusesand determines immune status of patients

•Assay Concept

•Introduction to nCounter System

•Some results of current work


3

• A major issue related to any large outbreak of infectious disease is rapid triage of those who are sick from the “worried well” who will rapidly overwhelm the healthcare system

• Most infectious diseases have similar initial symptoms:• Fever, generalized or targeted pain, lethargy, etc.

• However, health outcomes after infection by a new and deadly strain of flu or Ebola are dramatically different than those from more common pathogens

• A relatively rapid, simple, and accurate method for simultaneous detection of multiple targeted pathogens combined with simultaneous immune system profiling of each patient should enable rapid triage.


4

1. During a major outbreak, patients are directed to an isolated section of the hospital and give a small amount (5ml) of blood and sputum

2. Patients are then told to go home, isolate themselves from others, and wait for results

3. Patients are contacted within 24 hours with one of 3 possible diagnoses:

A. “You are fine”None of the agents detected and no elevation of immune response

B. “You are ill, but not suffering from the deadly outbreak”None of the agents detected, but immune response genes are elevated

C. “You are infected and should return to the hospital ASAP”One of the agents targeted is present in one or more samples and immune response genes are elevated indicating disease

A similar concept should also enable vastly improved primary care for patients outside the scope of a public health emergency


A technology platform to deliver highly multiplexed, direct profiling of individual molecules in a single reaction without amplification.

Slide Courtesy of Nanostring Corp.


NanoString enables sensitive analysis of hundreds of genes in a single‐tube assay

QPCR

Microarrays, NextGen Seq

Sens

itivity

Multiplexing Capability # of Transcripts

NanoString

6



Enables simultaneous measurement of 20 to over 900 targets per sample

Digital Detection and Analysis

No enzymatic reaction for Signal Amplification is Required; thus simplifying assay process

System ease of use including automated liquid handling

Simplicity of data analysis

Highly Sensitive, Reproducible Data

Flexibility in Sample Type

Over 81,000 Data Points in 24 hours

7


8

Biotin

Target Specific Capture Probe Target Specific

Reporter Probe



9

Target Specific Capture Probe

Target Specific Reporter Probe



Sequence‐Specific Capture Probes, Reporter Probes and System Controls are Combined to Create Custom CodeSet

0

100

200

300

400

500

600

0 20 40 60 80 100 120

Capture Probes

Reporter Probes

System Controls



Day2AUTOMATED

5 minHANDS‐ON

5 minHANDS‐ON

Day2AUTOMATED

5 minHANDS‐ON

5 minHANDS‐ON

Day15minHANDS ‐ON

5minHANDS ‐ON

PurifyPurify2HybridizeHybridize1 CountCount3



Removeexcessreporters

Bindreporterto surface

Immobilize and align reporter

Image surface

HybridizeCodeSet to RNA

Count codes

mRNA Capture & Reporter Probes Slide Courtesy of Nanostring Corp.





Image surface


Count codes

Hybridized mRNA Excess ReportersSlide Courtesy of Nanostring Corp.





Image surface


Count codes

Hybridized Probes Bind to Cartridge

Surface of cartridge is coated with streptavidin






Image surface


Count codes

Immobilize and align reporter for image collecting and barcode countingSlide Courtesy of Nanostring Corp.





Image surface


Count codes

Image Surface

One reporter code = 1 mRNA/viral RNA/miRNASlide Courtesy of Nanostring Corp.


Code Gene Countx 3

y 1

z 2

Codes are counted and tabulated




Image surface


Count codes



Analysis using Excel™



n=47 n=235 n=938n=778

n=173n=789Measurements are precise even at low transcript abundances



20

Total RNA (100ng/sample)

Amplified RNA from Small Amount of Sample

Whole Cell Lysates

PaxGene LysedWhole Blood

Total RNA Extracted from FFPE Samples

Crude Extracts from FFPE samples



21

LLNL:• Generate Virus‐Specific Signatures for Detection and Sub‐typing of 19 Category A,

B, and C Viral Agents• Amenable to nCounter Platform (150 to 350bp regions)• Amenable to TaqManAssay Development (60 to 200bp regions)

• Develop TaqManAssays Targeting Viral Agents• Perform nCounter Assays (Virus‐specific and Inflammation Panels)• Test and Compare Assay Sensitivity and Selectivity of TaqMan vs. nCounter for

Virus‐specific Signatures

UTMB:• Generate Viral Templates in Cell Culture and Mice and Send RNA to LLNL• Test Mouse Cytokine/Chemokine Responses Using Luminex Protein Assay

UCSF:• Provide Over 200 Clinical Samples from Human Infections• Perform Microarray and Sequencing on Samples and Compare Results with

Nanostring


22

BSL 2/BSL3 Viruses

BSL 4 Viruses

Dengue [1-4] Junin [Romero, XJ13] Ebola [Zaire, Reston, Sudan, IC]

West Nile [NY 99] Lassa [Josiah] Marburg [Ravn, Angola]Yellow Fever [Asibi] Machupo [Carvallo] Nipah [Malaysia]CHIK LR 2006-OPY1 Guanarito Hendra

Kyasanur Forest Disease CCHFV [IbAr10200]Russian Spring/Summer Enceph. [Sofjin] RVFV [ZH501, ZH548]Omsk Hemorrhagic Fever [Guriev]


100 respiratory outbreak specimens (California) – nasal / throat swabs

300 respiratory outbreak specimens (Canada) – nasal / throat swabs

400 diarrheal outbreak specimens (California) ‐ stool 500 diarrheal outbreak specimens (Canada) – stool 100 diarrheal outbreak specimens (Mexico) – stool 100 age‐, sex‐matched control specimens (Mexico, California) – respiratory secretions, stool

100 febrile hemorrhagic pneumonia and sepsis (India) ‐‐serum, respiratory secretions

100 outbreaks of febrile illness (Africa) – serum

23


24

• All Viruses to be Tested Have Been Generated at UTMB and Received as TrizolExtracts

• Designs for all TaqManAssays to be Developed are Complete and Testing of Cultured Samples has Begun

• Identification of all Viral Sequence Regions to be Targeted with nCounterCodesets is Complete and Testing Using the “Virus Counter” Codeset Has Begun

• Received Human “Inflammation Panel” nCounter Codeset (183 genes targeted)

• Received Mouse “Inflammation Panel” nCounter Codeset (178 genes targeted)

• Samples Collected at UCSF and Testing Using “Green Chip” and Sequencing Begun


25

• Collaboration between LLNL and USAMRIID (Dr. Sina Bavari)

• Determine miRNA expression changes in multiple tissues upon infection with Ebola Virus

• Identify novel miRNAs associated with Ebola infection in Cynomolousmacaques

•Utilizing the 745‐plex Human miRNA profiling panel


26

• Coverage of most human miRNAs in the miRBase v14.

• miRNA detection from 100ng total RNA isolated from blood, cell lines, fresh/frozen and FFPE tissue

• Highly sensitive: lower limit of detection comparable to other technologies

• Specific, single tube assay that distinguishes single nucleotide differences between miRNAs with minimal cross-hybridization


27

miRNAs Differentially Expressed in Naïve vs Ebola Virus‐infected Cynomolgus macaque Kidney

Naïve vs Ebola Virus-infected Cynomolgus Monkey Kidney Samples

Naïve Ebola-virus infected Significance Potential Targetshsa-miRNA* ID

Avg. Count St Dev

Avg. Count St Dev P-value Fold Change

hsa-miR-155 165.48 34.99 463.38 23.24 0.0005 2.80Targets Angiotensin II Type 1 receptor (hAT1R). Also targets

KGF 3'-UTR, CYR61, and rs5186 binding site in AGTR1 hsa-miR-484 292.32 20.61 546.29 8.02 0.0007 1.87 Targets not availablehsa-miR-375 2.82 N/A 48.28 4.23 0.0009 17.11 Targets neuronal RNA-binding protein HuDhsa-miR-301a 194.84 19.63 72.53 19.07 0.0015 -2.69 Regulates ska2hsa-miR-378 323.97 32.52 768.61 62.72 0.0016 2.37 Regulates caspase-3 activity

hsa-miR-199a-5p 781.99 78.27 304.12 115.65 0.0061 -2.57

Targets HES1, calcineurin/NFAT gene, the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation

regulated kinase 1a hsa-miR-26b 3221.82 197.26 5999.48 570.94 0.0080 1.86 Targets VEGF mRNA

hsa-miR-205 365.04 70.28 1.44 1.21 0.0085 -254.00 Regulates kinase Cepsilon, E2F1

*Homo sapien microRNA

Only miRNA expression pattern changes with P-value of 10-3 or better are reported. An additional 40+miRNAs with lower P-values have been identified to be differentially expressed.


0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00NaïveSamples(n=3)

Ebola Virus-infectedSamples(n=3)

26b 155 301a 375hsa-miRNA ID

Avg.

Cou

nts

Homo sapien miRNA (hsa-miRNA) Counts: Naïve vs Ebola Virus-infected Cynomolgus MonkeyKidney Samples

199a-5p 205378 484

1.86

-2.57

2.37 1.87

-254.00

2.80

-2.69

17.11

‐260.00

‐210.00

‐160.00

‐110.00

‐60.00

‐10.00

40.00

Naïve (n=3)vs. EbolaVirus-infected(n=3)

26b 155 301a 375

hsa-miRNA ID

Fold

Cha

nges

Homo sapien miRNA (hsa-miRNA) Fold Changes: Naïve vs Ebola Virus-infected Cynomolgus MonkeyKidney Samples

199a-5p 205378 484

P-value< 0.01

P-value< 0.01

miR 205 Expression is Essentially Eliminated in Ebola‐infected Kidney Samples


Naïve vs Ebola Virus-infected Cynomolgus Monkey Lungs Samples

NaïveEbola Virus-

infected Significance Potential Targets

hsa-miRNA* IDAvg.

Count St DevAvg.

Count St Dev P-valueFold

Changehsa-miR-1246 513.14 292.79 2802.01 405.45 0.0020 5.46 Targets not available

hsa-miR-21 9052.59 2241.91 37843.91 4943.09 0.0036 4.18 Targets HIV-1 3'UTR region

hsa-miR-155 235.20 148.53 1026.41 174.00 0.0042 4.36Targets 3'-UTR of human Angiotensin II Type 1 receptor(hAT1R).

Also targets KGF 3'-UTR, CYR61, and rs5186 binding site in AGTR1hsa-miR-223 2921.83 1959.20 16360.15 3266.12 0.0068 5.60 Targets not available

hsa-miR-125a-5p 1773.60 149.98 1188.98 131.92 0.0074 -1.49 Targets not available

*Homo sapien microRNA

Only miRNA expression pattern changes with P-value of 10-3 or better are reported. An additional 40+miRNAs with lower P-values have been identified to be differentially expressed.

Naïve vs Ebola Virus‐infected Cynomolgusmacaque Lung Samples


30

0.005000.00

10000.0015000.0020000.0025000.0030000.0035000.0040000.0045000.00

Naïve Samples(n=3)

Ebola Virus-infected Samples(n=3)

21 223 125a-5p 1246

Avg.

Cou

nts

155

Homo sapien miRNA (hsa-miRNA) Counts: Naïve vs Ebola Virus-infected Cynomolgus MonkeyLung Samples

4.18

5.60

-1.49

5.464.36

-8.00

-6.00

-4.00

-2.00

0.00

2.00

4.00

6.00

8.00

Naïve (n=3) vs.Ebola Virus-infected (n=3)

hsa-miRNA ID

Fold

Cha

nges

Homo sapien miRNA (hsa-miRNA) Fold Changes: Naïve vs Ebola Virus-infected Cynomolgus MonkeyLung Samples

21 223 125a-5p 1246 155

P-value < 0.01

P-value < 0.01

Most miRNAs are Up‐Regulated in Ebola‐infected Lung samples


31

•MiRNA levels from kidney, liver, pancreas and spleen samples of uninfected and Ebola‐virus infected Cynomolgus macaques, miRNAs potentially associated with Filovirus infection were identified:

•Kidney: 8 different miRNAs, including miR205 (our best potential target)•Liver: 9 different miRNAs•Pancreas: 10 different miRNAs•Spleen: 13 different miRNAs•Lung: 5 different miRNAs

•None of the tissues assayed had expression changes in the same miRNAs (those with P<0.01)

• In the kidney and liver samples, the majority of miRNAs showed an increase in expression post‐infection. In the pancreas and spleen samples, the majority of hsa‐miRNAs showed a decrease in expression post‐infection

• Several target miRNAs, like hsa‐miRNA 205, showed a dramatic change in miRNA expression and a significant fold change as compared to healthy samples

•Assays conducted on peripheral plasma have provisionally identified 3 miRNAs that may be of utility in differentiating an infected from a healthy individual

Preliminary Conclusions


Application/Work Still Pending•Illumina data from small RNA fraction has been generated for the same samples and is being analyzed

•Potentially novel miRNAs of monkey origin may have been found•The search continues for miRNAs of filovirus origin

•Samples from peripheral plasma at multiple days post‐infection from multiple animals are now being analyzed

•MiRNA 205 may be a good target for diagnostic development via two different routes:

•Develop an assay looking for this miRNA directly from urine•Look at the downstream targets of this miRNA and develop an assay targeting them or the small molecules that they may be generating


33

1. Identified multiple host miRNAs that appear to be affected by Ebola Virus infection and are thus potential targets for therapeutics development.

2. Technologies already being deployed by USAMRIID with DTRA funding that have shown efficacy targeting Ebola viral RNA directly can also be used to target host factors that may play a role in progression of infection.

3. The same process developed here should be applicable to any number of other viruses.

So What?


34

DTRA CB Diagnostics and NIAID for Funding

Nanostring Corp.: Dr. Steve Jackson

UCSF: Dr. Charles Chiu

UTMB: Drs. Alex Freiburg and David Beasley

LLNL: Dr. Shea Gardner, Celena Carrillo, Ana Rosa, Sarah Hall, Maher Elsheikh, Sanaz Mohammadi

pejman naraghi arani, phd - aacc

Documents