Confidential

Extending the Armored RNA Technology to DNA

Cindy WalkerPeach, Ph.D.Director, Development and Operations

Ambion Diagnostics

BSI EN ISO9001:2000 CertifiedBSI Registration Number: FM 57081

Quality System Regulations (21CFR§820)Medical Device Manufacturing

Owner/Operator Number: 9049048

Confidential

Armored RNA -> Armored RNA QuantPackaging System

Armored RNA®

Armored RNA Quant™

Confidential

Armored DNA Proof of Principle – Concept

Armored DNA

Armored RNA Quant™Armored DNA

Confidential

DNA Controls & StandardsCommonly Used

• DNA plasmids

• Infectious or attenuated / inactivated viruses

• Patient samples

• PCR amplicons

Confidential

Armored DNA Proof of Principle – DNA Packaging of two Targets

Packaging of two unrelated sequences900bp

Armored D

NA

Armroed RNA control

Ladder

239bp PCR product

Armored D

NA

Armroed RNA control

Ladder

371bp PCR product

900bp

Confidential

Armored DNA Proof of Principle - Resistant to DNase

1 ug of PCR target or 5 ug of Armored DNA Internal Control was treated with and without Turbo DNase for 1 hour

  DNA DNA Marker Armored Armored

DNA DNANo DNase +DNase No DNase +DNase

Confidential

Armored DNA Proof of Principle – DNA Protection

DNA protected from nuclease degradation

AD (Packaged,Extracted)

Extracted DNA fragm

ent

DNA fragment

Ladder

239bpAD (Packaged,Extracted) Extracted DNA fragm

entDNA fragm

entLadder

371bp

Confidential

Armored DNAProof of Principle - QC

ARQ Phosphate Standard Curve

y = 0.0171x + 0.2215R2 = 0.9984

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20 25 30 35 40 45

ng Phosphate

OD2

60

Phosphate Assay HPLC Assay

DNA Integrity Functional Testing (PCR)

Confidential

Armored DNA Proof of Principle – Quantification

 

Phosphate Assay

(copies/uL)

qRT-PCR HBV Assay

(copies/uL)

HBV 8.0 x1010 (±0.65 x 1010)

8.6 x1010

7% Difference

Confidential

Armored DNA Heat Stability Profile AD Internal Control

Control 50 C 60 C 70C ARQ Armored DNA ARQ Armored DNA ARQ Armored DNA ARQ Armored DNA

Confidential

Armored DNA Detection Over a Linear Range

Syber Green Assay of Armored DNA HBV

y = -1.6067Ln(x) + 18.27R2 = 0.9993

y = -1.5763Ln(x) + 18.055R2 = 0.9998

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30.00

35.00

0.001 0.01 0.1 1 10 100

Armored DNA Input (picograms)

Ave

Ct

Log. (Primer pair B)

Log. (Primer pair A)

Parallel assays of AD HBV serial curve (42 to 0.042 pg)Test with two sets of primersDirect input of samples, 2 ul per reaction in duplicate

Confidential

Armored DNA Initial Stability in Plasma

4 copies/uL (AD HBV 239bp) incubated in plasma(1 x 103 copies extracted in duplicates)

• Stable 7 days at 37°C• Stable 14 days at 25°C• Stable 1x freeze-thaw• CV < 4%

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0.50

1.00

1.50

2.00

2.50

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3.50

4.00

Baseline 2 days, 37C 7 days, 37C 7 days, 25C 14 days, 25C 1X Freeze-Thaw

Log

copi

es/rx

n

Confidential

Armored Technology Armored DNA summary

• Armored Technology compatible with DNA • Packaging of 239 and 371bp double stranded DNA up to an

undefined limit• Stable in Plasma

— 7 days at 37°C— 14 days at 25°C— 1x freeze-thaw

• Stable in TSMIII— 7 days at 37°C — 7 days at 25°C — 14 days at 4°C — 1x freeze-thaw

Confidential

Armored DNA Ideal Controls and Standards

• Stable – Nuclease resistant• Quantifiable – Armored DNA referenced to a NIST

standard• Homogeneous – single, sequence defined target• Compatible – compatible with broad range of assay

technologies• Flexible – allows for addition to human matrix of

choice• Customizable – user defined sequences, genotyping• Non-infectious – ease in handling and shipping