Ando Sangamo

Endpoints
in HIV
• Viral Load
– Accepted Regulatory Endpoint in Viremic Subjects
– Not useful in Aviremic subjects. Treatment Interrup@on viral set point in use in
immunologic and cell therapy clinical trials for HAART treated subjects.
• Proviral DNA
– Novel endpoint with unknown clinical significance.
– Complicated by non-‐func@onal proviral DNA versus func@onal
– Methyla@on status of LTR maybe important
• Reservoir Analysis for HIV
– Novel endpoint with unknown clinical significance.
– Include PCR quan@fica@on, viral co-‐culture etc
• CD4 T cells
• Clinical trials using AIDs endpoints are large and long-‐term (SILCAAT)
• Total number of CD4 and HIV specificity are being evaluated in HIV e.gl IL-‐7, Therapeu@c
vaccines
• Advances in Digital PCR and Deep sequencing are poten@al breakthrough
technologies in the future.
1. Digital Droplet PCR -‐ Principles
Step 1. “Digi@ze” sample into ~15,000

droplets, effec@vely reducing level
of background gDNA
Step 2. Run PCR to endpoint. Quan@fica@on

no longer relies on PCR kine@cs
Step 3. Fluorescent analysis drop by drop

(yes or no). Copy number calculated
by Poisson distribu@on
2. Digital Droplet PCR Allows for More Precise
QuanCtaCon of Low Copy Number Events
• Reduce genomic DNA inhibiCon
• For many HIV+ subjects, the HIV-‐DNA copy number per cell can be very low (0.1
to 0.01% of cells)
• Quan@ta@on of HIV DNA copy number by tradi@onal qPCR o_en experienced
inhibi@on due to the low ra@o of HIV templates to background genomic DNA
• By separa@ng template DNA into > 10,000 droplets, the posi@ve template (HIV
DNA) to background DNA (genomic DNA) ra@o is increased by 4 logs
• Improve precision with digital PCR

• With digital PCR, reac@on is carried out to comple@on (endpoint).
Quan@fica@on is no longer dependent on PCR kine@cs. PCR standard is NOT
needed.
• Concentra@on of templates is calculated by the number of posi@ve and
nega@ve events (hence digital) using Poisson analysis
3. Digital Droplet PCR – Experimental Methods (1)
1. Digest genomic DNA sample with DdeI

(37oC, 1 Hr)
2. Prepare mulCplex PCR Master mix with
primer/ probes:
• HIV-‐gag (FAM)
• Primer/ probe as described in Palmer et. al., JCM
2003
• RNaseP (VIC)
3. Prepare 2X PCR reacCons, each with 500µg
and 250µg gDNA (1.5mg total gDNA)
4. Generate droplets (figure right)
5. Perform convenConal PCR

Thermal Cycling Parameters Cycles Duration
95°C 1 10 min
30 sec (melt)
94°C melt and 60°C anneal/extend 40
1 min (anneal/extend)
98°C Heat inactiviation 1 10 min
4°C hold 1 indefinite

4. Digital Droplet PCR – Experimental Methods (2)
6. Analyze droplets for +/-‐ FAM and VIC

fluorescent signal (figure right)
7. Copy number is determined by Poisson
analysis (figure below)
5. Precise DeterminaCon of HIV-‐DNA Copy Numbers
“High” HIV-‐DNA level “Low” HIV-‐DNA level

(2.5% of cells) (0.05-‐0.1% of cells)
• Mean = 26562 copies/1x106 cells • Intra-‐assay • Inter-‐assay

• Std Devia@on = 2076 • %CV = 18% • %CV = 22%
• %CV = 7.8%
6. Assessing the QuanCtaCve Limits of the HIV-‐DNA DD
PCR Assay
HIV+ Sample 1 HIV+ Sample 2
HIV-‐DNA copies/ 1x10^6 PBMC 1024
512
256
[Log 2 Scale]
128
64
32
16
8
Undiluted 1:2 1:4 1:8 1:16 1:32
• HIV+ gDNA samples were serially diluted with normal human liver gDNA (2-‐fold dilu@on)
• In two independent HIV+ gDNA samples evaluated, 2-‐fold dilu@onal linearity was
observed down to 0.01% (or ~100 copies per 1x10^6 cells)
8. Digital Droplet PCR Outperforms ConvenConal Taqman
qPCR
HIV-DNA/ 1e6 PBMC

DD PCR Conventional Taqman
• Conven@onal Taqman qPCR performed
Sample 1 332 Undetected as described in Desire et. al., J. Clin.
Sample 2 396 Undetected
Micro., Apr 2001, p.1303
Sample 5 323 Undetected • 24 PBMC samples obtained from HIV+
Sample 7 168 Undetected subjects were evaluated with both DD
Sample 8 165 Undetected PCR and conven@onal Taqman:
Sample 11 709 Undetected • HIV-‐DNA detected in all samples
Sample 13 443 Undetected with DD PCR (range = 12 to 3300
Sample 14 339 Undetected copies per 1x106 cells)
Sample 17 27 Undetected • Conven@onal Taqman detected
Sample 19 12 Undetected HIV-‐DNA in only 2 samples
Sample 20 53 Undetected (samples with the highest HIV
Sample 21 3250 192
Sample 22 3308 158 copy numbers according to DD
Sample 23 1430 Undetected PCR, boxed in red)

Ando Sangamo

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Ando Sangamo

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Endpoints

Step 1. “Digi@ze” sample into ~15,000

Step 2. Run PCR to endpoint. Quan@ﬁca@on

Step 3. Fluorescent analysis drop by drop

• Improve precision with digital PCR

1. Digest genomic DNA sample with DdeI

4°C hold 1 indefinite

6. Analyze droplets for +/-‐ FAM and VIC

“High” HIV-‐DNA level “Low” HIV-‐DNA level

• Mean = 26562 copies/1x106 cells • Intra-‐assay • Inter-‐assay

HIV-DNA/ 1e6 PBMC

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Ando Sangamo

Uploaded by

Ando Sangamo

Uploaded by

Endpoints

Step 1. “Digi@ze” sample into ~15,000

Step 2. Run PCR to endpoint. Quan@ﬁca@on

Step 3. Fluorescent analysis drop by drop

• Improve precision with digital PCR

1. Digest genomic DNA sample with DdeI

4°C hold 1 indefinite

6. Analyze droplets for +/-­‐ FAM and VIC

“High” HIV-­‐DNA level “Low” HIV-­‐DNA level

• Mean = 26562 copies/1x106 cells • Intra-­‐assay • Inter-­‐assay

HIV-DNA/ 1e6 PBMC

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6. Analyze droplets for +/-‐ FAM and VIC

“High” HIV-‐DNA level “Low” HIV-‐DNA level

• Mean = 26562 copies/1x106 cells • Intra-‐assay • Inter-‐assay