Abstract # 874

METHOD DEVELOPMENT AND VALIDATION FOR qPCR ANALYSIS OF MALE PORCINE CELL BIODISTRIBUTION IN FEMALE PIGS Maoxiang Li1, Emily Hill1, Jeffrey Grover1, Valerie Steenwinckel2, Mark Wolfe1, Mark Johnson1 and Haiyan Ma1
1. Department of Cellular and Molecular Biology, MPI Research Inc., U.S.A. 2. Cardio3 Biosciences S.A., Belgium

May 18, 2012

INTRODUCTION
Stem cells, whether gene-modified or not, present a number of safety concerns after administration into human subjects. Major questions have been raised as to where stem cells migrate to after administration, whether the cells engraft and differentiate, and what their long-term fate is. Therefore, GLP-compliant preclinical biodistribution and persistence animal studies are recommended by the United States Food and Drug Administration (FDA) to evaluate the safety of potential stem cell-based products. Quantitative PCR is currently the most sensitive detection method for biodistribution of cell and gene therapy products. For this purpose, a quantitative real time polymerase chain reaction (qPCR) method for biodistribution analysis of male mesenchymal stem cells (MSC) in female pig tissues has been developed and validated. In this study, the specificity of the TaqMan probe/primers to the male porcine sex-determining region Y (SRY) gene sequence was determined by carrying out real-time PCR on genomic DNA isolated from male porcine MSC in the absence and presence of female pig matrix genomic DNA (gDNA). The qPCR analysis was optimized. The low limit of detection (LLOD), low limit of quantification (LLOQ), upper limit of quantification (ULOQ), and the potential effects of female porcine matrix gDNA were evaluated by conducting a series of qPCR experiments on total female pig DNA spiked with known amounts of male MSC DNA. The PCR efficiency was estimated through the linear regression of the dilution curve. The intra-assay precision, inter-assay reproducibility, and accuracy were evaluated over five separate assays. For each assay, two sets of quality control (QC) samples were prepared independently and each set included three concentrations of male pig MSC DNA (high, middle and low) in triplicate. The assay specificity, linear range, intra- and inter-assay precision, accuracy and reproducibility, and acceptance criteria are reported.

RESULTS
Method Specificity:
Sample QC-low QC-mid QC-high

Quantity (copies/reaction) Nominal 100 1,000 28,500 Measured 108 989 28,358 % of Nominal 108 99 100

Table 4. Assay accuracy. The accuracy of the assay was evaluated by comparing the measured mean quantities of the ten sets of QC samples from the five assays with their expected nominal values. Table 5. Inter-assay precision. The mean, SD, and percent CV at each standard concentration were calculated across all reactions performed in the five runs of the qPCR assay.

Standards
(copies/reaction)

Positive Reaction 15/15 15/15 15/15 15/15 15/15

Ct Mean 21.99 25.34 30.43 34.06 35.13

Ct SD 0.084 0.094 0.116 0.234 0.272

Ct %CV 0.4 0.4 0.4 0.7 0.8

285,000 28,500 1000 100 50

OBJECTIVES
To develop and validate a method for detection of male MSC biodistribution in female pig tissues using the Applied Biosystems 7900HT Fast Real Time PCR system. The validated method will be used in GLP compliant studies in MPI Research.

Figure 1. Representative Amplification Plot of qPCR. The released reporter fluorescence (y-axis) from qPCR of male porcine MSC gDNA is plotted as a function of the amplification cycle number (xaxis). Serial dilutions of porcine gDNA copies in triplicate are shown (285000, 28500, 1000, 100, 50 and 0 copies/reaction), from left to right.

Figure 2. Specificity of Primers/Probe for Male Porcine MSC DNA. The negative control with 0 copies/reaction of male porcine MSC gDNA shows no amplification of the female porcine gDNA (present in this reaction at 1000, 500, 200, and 0 ng/reaction), demonstrating the specificity of this assay to male porcine gDNA.

Assay Date Slope E Y-intercept R2

A1 08/31/11 -3.537 0.918 41.226 0.999

MSC qPCR for Biodistribution A2 A3 A4 09/01/11 09/02/11 09/06/11 -3.532 -3.496 -3.495 0.919 0.932 0.932 41.134 41.006 40.981 0.997 0.998 0.997

A5 09/07/11 -3.428 0.958 40.648 0.998

Table 6. Standard curve best-fit analysis. The slope, efficiency (E), Y-intercept, and R2 value for each plate run were compared across five runs of the qPCR assay.

Linearity and Method Sensitivity:

Standard Concentration
(copies/reaction)

Table 7. Summary of Validation Results:

Test Individual Accuracy (QC) 45% RE 45% CV (Quantity) 2% CV (Ct) 45% RE 45% CV Quantity) 3% CV (Ct) 2% CV (Ct) (For 285,000 50 copies male MSC gDNA/reaction) Acceptance Criteria Results -22.2% to 38.2% 20.7% CV (Quantity) 0.9% CV (Ct) -1.1% to 8.0% 15.2% CV (Quantity) 0.7% CV (Ct) 0.8% CV (Ct) 50 to 285,000 copies of male porcine MSC gDNA/ mg of total DNA in reaction R2 0.95 80% E 110% Two out of three replicates from each assay have a percent CV (Ct) 2%; all replicates from 5 assays show positive in qPCR Two out of three replicates from each assay have a percent CV (Ct) 2%; all replicates from 5 assays show positive in qPCR Detected in at least two of the three replicates 0.997 92% - 96% 50 copies male porcine MSC gDNA / mg of total DNA / reaction 285,000 copies male porcine MSC gDNA / mg of total DNA 5 copies male porcine MSC gDNA / mg of total DNA / reaction

Positive Wells/Total Wells per Assay A1 A2 A3 A4 A5 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 1/3 2/3 2/3 2/3 1/3 1/3 1/3 0/3 1/3 1/3 0/3 0/3 0/3 0/3 0/3 5 2.85 x 105 2.85 x 105 2.85 x 105 2.85 x 105 2.85 x 10 50 10 20 5 5 5 2 2 2 5 0.953 0.943 0.932 0.969 0.988

Intra-assay Precision (QC) Over-all Accuracy (QC) Inter-assay Precision (QC) Inter-assay Precision (Standards) Range Linearity (Goodness-of-Fit) Amplification Efficiency LLOQ Determination ULOQ Determination

METHODOLOGY
The study was based on current International Conference on Harmonisation (ICH) Harmonised Tripartite Guidelines1 and Guidance for Industry Gene Therapy Clinical Trials Observing Subjects for Delayed Adverse Events2 and conducted in accordance with the United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations, 21 CFR Part 58. The test article DNA was purified from male porcine MSCs using the QIAcube instrument with the QIAGEN QIAamp DNA mini kit. Multiple DNA mini-preps were pooled to create the standard target DNA. DNA concentration and quality were determined with the Nanodrop 8000 spectrophotometer. Female porcine matrix DNA was prepared from female swine liver tissue using the BioRobot M48 instrument and MagAttract M48 DNA mini kits. Equal volumes of multiple mini-preps were pooled and used as matrix DNA. DNA concentration and quality were determined with the Nanodrop 8000 spectrophotometer. The standard curve was constructed with serial dilutions of male porcine MSC gDNA to 2.85 x 105, 2.85 x 104, 103, 102, 50, 20, 10, 5, 2, 1 and 0 copies per 50 l of reaction. The QCs were prepared using male porcine MSC gDNA at nominal 102 copies (QC-low), 103 copies (QC-mid), and 2.85 x 104 copies (QC-high) per 50 l reaction. Each concentration was prepared in triplicate. If the male porcine MSC gDNA concentration was less than 1000 ng/reaction/well, female matrix gDNA was added to the well to make total DNA of 1g/well. Primers and TaqMan probe for the SRY gene were synthesized by Applied Biosystems. A series of primer/probe concentrations were combined and tested by qPCR and the set of primers/probe concentrations which yielded low Ct and %CV was chosen for this study.
SRY-R3 Ct Mean Ct SD Ct%CV Ct Mean Ct SD Ct%CV Ct Mean Ct SD Ct%CV Ct Mean Ct SD Ct%CV SRY-P3 33.0618 0.0508 0.1537 32.9678 0.0407 0.1234 33.0646 0.0194 0.0586 32.8600 0.1003 0.3053 300nM 900nM 33.1207 0.1538 0.4644 33.2362 0.0090 0.0272 33.2567 0.1997 0.6005 33.0073 0.0721 0.2183 200nM 600nM 33.2198 0.1300 0.3913 33.2309 0.1534 0.4615 33.2742 0.1671 0.5023 33.1405 0.2095 0.6321 100nM 300nM 33.6778 0.0834 0.2475 33.5371 0.2506 0.7471 33.4025 0.1764 0.5280 33.3461 0.0654 0.1961 50nM 100nM 900 nM 600 nM 300 nM 100 nM 100 nM Ct Mean Ct SD Ct%CV 32.7921 0.1135 0.3461 33.1135 0.2541 0.7675 33.8028 0.1377 0.4074 35.0802 0.1285 0.3662

285,000 28,500 1000 100 50 20 10 5 2 1 0 ULOQ/Assay LLOQ/Assay LOD/Assay Amplification Efficiency

Table 1 Sensitivity Detections. Five separate assays were completed with serial dilutions of male porcine MSC gDNA copies in female matrix DNA up to total DNA 1000 ng/reaction in triplicate. The LLOQ was set at 50 copies/reaction.

Figure 3 Representative Standard Plot. Standard points are shown from right to left, 285,000, 28,500, 1000, 100, and 50 copies/reaction.

LOD Determination

Precision, Accuracy and Reproducibility of Intra- and Inter-assays:

Validation Run A1 Sample QC-low QC-mid QC-high QC-low QC-mid QC-high QC-low QC-mid QC-high QC-low QC-mid QC-high QC-low QC-mid QC-high No. of Reactions 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Quantity (copies/reaction) Mean SD CV (%) 110 14.6 13.3 1074 31.0 2.9 28197 1427.4 5.1 108 14.3 13.3 1012 59.1 5.8 26957 2227.5 8.3 119 11.4 9.6 1000 90.1 9.0 27534 1191.1 4.3 97 14.6 15.1 878 33.1 3.8 30183 1150.4 3.8 107 22.2 20.7 982 83.4 8.5 28919 2036.2 7.0

CONCLUSIONS
A TaqMan based qPCR assay has been developed and validated to detect and quantify male MSC biodistribution in female pig tissues. The assay is specific for male porcine MSC gDNA and does not amplify female porcine genomic DNA. Lower Limit of Detection (LOD): 5 copies male porcine MSC gDNA / g total DNA Lower Limit of Quantification (LLOQ): 50 copies male porcine MSC gDNA / g total DNA Upper Limit of Quantification (ULOQ): 285,000 copies male porcine MSC gDNA / g total DNA Assuming male and female swine cells contain roughly equal amount of gDNA, the linear detection range of male swine cells in the background of female swine cells ranges from 0.0175% to 100%, and the limit of detection is 0.00175%. The precision, accuracy and reproducibility of the assay were evaluated and found to be suitable for quantifying male porcine MSC gDNA in DNA samples isolated from female porcine tissues.

A2

SRY-F3 / 900 nM SRY-R3

SRY-F3

A3

Each qPCR was conducted at 50 mL/well on a 96-well plate on a validated 7900HT Fast Real Time PCR system.

A4

qPCR Components in 50 mL 2x TaqMan Universal Master mix Forward primer SRY-F3 Reverse primer SRY-R3 TaqMan Probe SRY-P3 Total DNA (target & matrix) H 2O 25 mL (1X) 100 nM 900 nM 300 nM 1000 ng to final volume of 50 mL

qPCR Protocol
Hold Hold 50C 95C 2 minutes 10 minutes

Table 2. Intra-assay precision. Five runs of the qPCR assay (A1 to A5) were performed across five days with independent preparations of a standard curve and two sets of QC samples each day. Each set of QC samples included a high, middle, and low concentration of male porcine MSC gDNA across the linear range of the standard curve. The mean quantity of male porcine MSC gDNA, standard deviation (SD), and percent CV at each concentration of one assay run were calculated across all reactions performed on this concentration within the run.

A5

QC-low, 100 copies/reaction; QC-mid, 1,000 copies/reaction; QC-high, 28,500 copies/reaction

40 cycles 95C, 15 seconds; 60C, 1 minute

Sample QC-low QC-mid QC-high No. of Reactions 29 30 30 Quantity (pg/reaction) Mean 108 989 28358 SD 16.4 87.9 1923.5 CV (%) 15.2 8.9 6.8

REFERENCES & ACKNOWLEDGEMENTS

Table 3. Inter-assay precision. The mean, SD, and percent CV at each QC concentration were calculated across all reactions performed in the five runs of qPCR assay. 1. Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals. ICH M3 (M), Nov 9, 2000. 2. Guidance for Industry Gene Therapy Clinical Trials Observing Subjects for Delayed Adverse Events U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research, November 2006 We thanks Fanny Huberty, Sbastien Wynant and Sbastien Maun for their contribution to this work. Current study was supported by Cardio3 BioSciences and by La Region Wallonne grant 6363.

QC-low, 100 copies/reaction; QC-mid, 1,000 copies/reaction; QC-high, 28,500 copies/reaction