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  • 1.  PCR methodology for GTx

    Posted 01-11-2023 14:31
    I have a question of those who are working in the area of gene therapy, specifically related to PCR methods used for biodistribution, PK and shedding endpoints.

    There are multiple PCR technology platforms available to us, including "old school" real-time qPCR, newer digital drop PCR and newest chip-based dPCR. The newer platforms come with some advantages. My question is, which platform would you choose and why? Moreover, is there any concern about regulator's acceptance of data from newer platforms? Does anyone have experience submitting data generated by ddPCR or dPCR?

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    John Kamerud Ph.D.
    Director
    Pfizer Inc
    Andover MA
    [email protected]

    Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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  • 2.  RE: PCR methodology for GTx

    Posted 01-12-2023 08:20
    Hi John,
    We continue to use "old school" qPCR for biodistribution and vector shedding. The main reasons are that we have the instruments readily available, but also due to the higher dynamic range of qPCR as compared to ddPCR. The lower cost of qPCR is a factor as well. We use dPCR/ddPCR for determination of virus titer where a standard free and highly accurate measurement is truly required. - Lena

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    Lena Hofer
    Spark Therapeutics
    Philadelphia PA
    [email protected]

    Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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    Original Message


  • 3.  RE: PCR methodology for GTx

    Posted 01-13-2023 14:16
    Hi John,

    I agree with Lena and adding in my response that was posted in the AAPS community discussion board as well.

    Both traditional qPCR and dPCR (droplet or chip-based) have been used for several of the bioanalytical applications you listed. Either of these platforms can be used however there are some pros/cons to using one over the other depending on which application it is being used for. I tend to see biodistribution assays on traditional qPCR since it has a wider dynamic range than dPCR. The limited dynamic range on the top end for dPCR would potentially have implications in sample analysis for targets that are expected to have high copies and could lead to extra sample dilutions and retesting. I prefer to use dPCR for viral shedding assays because it has an advantage of better tolerance to PCR inhibitors and works well for matrices like feces, saliva, etc. dPCR tends to also be favored for cellular kinetics/persistence assays due to potentially better precision at the low end for sensitivity needs. That all said, I have seen qPCR and dPCR used for all of these applications and the decision ultimately depends on the target, matrix, and assay itself. These are just general observations between both platforms. Based on our discussions in the qPCR working group, I can't recall any instances where a dPCR was not accepted by a regulatory agency. 

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    Amanda Hays Ph.D.
    Scientific Officer
    BioAgilytix
    Olathe KS
    [email protected]

    Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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    Original Message