ddPCR in Pharmacokinetics: Revolutionizing Therapeutic Monitoring Across Advanced Therapeutic Modalities

Droplet digital PCR (ddPCR) is transforming pharmacokinetic (PK) monitoring across advanced therapeutic modalities, encompassing gene therapies, cell therapies, tissue-engineered products, and other nucleic acid–based medicines. Unlike conventional small-molecule drugs, these advanced therapeutics—including CAR-T cells, AAV-based gene therapies, antisense oligonucleotides (ASOs), and RNA-based drugs—are biologically complex, often personalized, and exhibit unconventional in vivo behavior that challenges traditional PK assessment. Regulatory frameworks such as those of the EMA and FDA classify gene and cell therapies as advanced therapy medicinal products (ATMPs), subdivided into gene therapy medicinal products, somatic cell therapy medicinal products, and tissue-engineered products, each presenting distinct requirements for biodistribution analysis, dose optimization, and long-term monitoring. Conventional analytical methods frequently lack the sensitivity and robustness needed to quantify low-abundance or transient therapeutic entities in complex biological matrices. By enabling Poisson-based absolute quantification, near–single-copy sensitivity, and high reproducibility without reliance on calibration standards, ddPCR provides a powerful solution for characterizing exposure–response relationships, tracking biodistribution and persistence, and supporting regulatory decision-making across the rapidly expanding landscape of advanced therapeutics.

Unlike quantitative PCR (qPCR), which depends on cycle threshold values and calibration standards for relative quantification, ddPCR partitions each reaction into tens of thousands of nanoliter-scale droplets, performs endpoint PCR in each discrete partition, and then applies Poisson statistics to calculate absolute target concentrations. This fundamental difference minimizes the impact of amplification efficiency variability and matrix-dependent inhibition, thereby improving reproducibility and comparability across studies. These characteristics make ddPCR particularly well suited to the complex PK characterization required for next-generation therapeutics, including viral vectors, cell and gene therapies, antisense oligonucleotides, gene-editing agents, and RNA-based drugs.

1.  VIRAL VECTOR-BASED GENE THERAPY

Fundamental challenges in viral vector pharmacokinetics

Viral vector-based gene therapies represent one of the most promising yet analytically challenging therapeutic modalities. Adeno-associated viruses (AAV), lentiviruses, oncolytic viruses, Boca virus, anellovectors, and adenoviruses function as delivery vehicles for therapeutic transgenes, but their pharmacokinetic behavior encompasses multiple interlinked processes: systemic distribution of vector particles, cellular uptake and transduction, episomal persistence or genomic integration, and the temporal profile of transgene expression. Traditional methods such as ELISA for capsid proteins or qPCR for vector genomes frequently lack the sensitivity, robustness, or dynamic range required to track these processes over time, particularly as vector concentrations fall to low levels during the terminal phase or in tissues with low transduction efficiency.

Applications of ddPCR  in preclinical toxicology studies of AAV-based gene therapies

In preclinical mouse and non-human primate (NHP) toxicology studies, ddPCR has become indispensable for quantitative characterization of vector biodistribution and shedding. Following systemic administration of AAV vectors, concentrations in plasma can span seven to eight orders of magnitude, from peak levels of approximately 10¹¹–10¹³ vector genomes (vg)/mL to late timepoints where only residual vector remains. The capacity of ddPCR to accurately quantify over this range,  and associated variability, is critical for defining dose, exposure relationships and establishing safety margins.

Tissue biodistribution studies in GLP toxicology protocols require quantification of vector genomes in numerous organs, often exhibiting markedly different levels of transduction. ddPCR assays targeting conserved inverted terminal repeat (ITR) sequences or transgene-specific regions enable absolute quantification of total vector genomes in homogenized tissues. More sophisticated assay designs can distinguish episomal from integrated forms, providing mechanistic insight into vector persistence and potential integration-related risks. In NHP studies for neurological gene therapies employing intrathecal or intracerebroventricular administration, ddPCR’s high sensitivity is especially valuable for detecting low-level vector presence in cerebrospinal fluid (CSF) and peripheral organs, thereby informing safety assessments around off-target transduction.

Transgene expression persistence represents another critical PK-related parameter for gene therapy. ddPCR assays directed at vector–host genome junctions (for integrating vectors) or at transgene-specific mRNA sequences (using RT-ddPCR) enable quantitative monitoring of transduction stability during chronic toxicology studies spanning months in NHPs. These data feed directly into human dose selection, dosing interval decisions, and long-term benefit–risk assessments.

Applications of ddPCR in clinical trials of AAV-based gene therapies

The transition from preclinical to clinical development introduces additional complexity in sampling matrices, regulatory expectations, and patient heterogeneity. In first-in-human Phase 1 dose-escalation trials, ddPCR provides the analytical sensitivity required to detect vector shedding in blood, urine, saliva, and semen, satisfying regulatory requirements for evaluating environmental release and transmission risk. The absolute quantification afforded by ddPCR removes dependence on matrix-matched standard curves, which can be challenging to establish for diverse bodily fluids.

In Phase 2 and 3 efficacy trials, ddPCR-based vector exposure metrics can be correlated with clinical outcomes. For systemic AAV therapies targeting liver (e.g., hemophilia and metabolic diseases), muscle (e.g., Duchenne muscular dystrophy), or the central nervous system (e.g., spinal muscular atrophy), quantification of vector genomes in circulating cell-free DNA or accessible tissues allows exposure–response modeling and supports refinement of dosing strategies.

A particular advantage of ddPCR emerges when investigating patients with pre-existing anti-AAV neutralizing antibodies, which can substantially alter vector pharmacokinetics. By quantifying vector genomes in blood and tissues, ddPCR can reveal whether antibody-bound vector complexes exhibit different distribution and clearance patterns compared with unbound vector, informing patient selection criteria, immunomodulatory strategies, and potential re-dosing paradigms.

Applications of ddPCR in commercialization and post-marketing surveillance of AAV-based gene therapies

Following regulatory approval, ddPCR continues to play crucial roles in manufacturing quality control and post-marketing surveillance. Vector titer determination by ddPCR provides robust lot-to-lot comparability with reduced inter-assay variability compared with qPCR-based methods, supporting consistency of commercial products. [5] Validated ddPCR assays also underpin pharmacovigilance programs that track long-term vector persistence and durability of transgene expression, key determinants of real-world effectiveness and the need for re-dosing.

Recent innovations include multiplex ddPCR assays capable of quantifying multiple vector serotypes in combination gene therapy strategies and highly sensitive assays designed to detect replication-competent AAV (rcAAV) or replication-competent lentivirus (RCL). These safety-critical assays must achieve extremely low detection limits, in line with regulatory expectations that rcAAV or RCL be absent or below stringent thresholds, often less than one infectious unit per intended clinical dose.

2.  CELL THERAPY PRODUCTS

CAR-T and TCR-T cell pharmacokinetics

Adoptive cell therapies, especially chimeric antigen receptor T cells (CAR-T) and T-cell receptor–engineered T cells (TCR-T), create unique pharmacokinetic challenges. Unlike conventional drugs exhibiting relatively predictable distribution and elimination, these “living drugs” undergo in vivo expansion, contraction, and long-term persistence phases influenced by tumor burden, host immunity, and construct design. ddPCR has emerged as a reference approach for characterizing these cellular kinetics, offering sensitivity and precision that complement and often surpass flow cytometric enumeration alone.

Applications of ddPCR in preclinical development in mouse and NHP models

In xenograft mouse models, ddPCR allows precise quantification of human CAR-T cells against a murine genomic background by targeting the CAR transgene or human-specific genomic regions. This specificity is crucial for distinguishing therapeutic cells from background cells, particularly as T-cell counts decline to low levels during the contraction phase. Assays can be configured to measure total CAR transgene copies, reflecting all transduced cells regardless of surface expression, or to estimate integrated vector copy number, providing insight into transgene silencing phenomena or clonal selection.

NHP studies for autologous cell therapies demand even more nuanced analytical strategies. Because both therapeutic and endogenous cells are of primate origin, ddPCR assays must target the transgene or unique sequences introduced during cell manufacturing. Multiplex ddPCR panels that quantify CAR transgene copies alongside reference genes enable determination of vector copy number per cell and facilitate differentiation between changes in total cell number versus changes in copy number per cell due to clonal expansion. Biodistribution studies examining off-tumor trafficking, a key safety concern, use ddPCR’s sensitivity to detect rare CAR-T cells in tissues such as brain, heart, and lung, where on-target/off-tumor toxicity could manifest. These data directly shape clinical risk mitigation strategies and monitoring plans.

ddPCR in clinical trial implementation of CAR-T cells

Phase 1 CAR-T trials typically employ ddPCR as a complementary PK tool alongside flow cytometry. Flow cytometry provides phenotypic information and surface CAR expression, whereas ddPCR delivers highly sensitive and robust quantification of CAR transgene copies, enabling detection of low-level persistence that may fall below flow cytometric detection thresholds.

Exposure–response analyses based on ddPCR data have been central to understanding both efficacy and toxicity. Higher peak CAR-T expansion is generally associated with improved clinical response but also with increased risk of cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS). By providing absolute transgene quantification independent of surface expression, ddPCR has revealed that some durable responders maintain very low yet detectable CAR-T levels for years, concentrations that may evade detection by flow cytometry yet remain pharmacologically relevant.

For allogeneic cell therapies, ddPCR enables discrimination between donor and recipient cells through single nucleotide polymorphism (SNP)-based assays or by targeting intentional genetic modifications such as TRAC gene disruption. This capability is essential for understanding persistence and rejection of allogeneic cells, as well as assessing the risk of graft-versus-host disease.

Applications of ddPCR in TIL and other cell therapy modalities

Tumor-infiltrating lymphocyte (TIL) therapies and other adoptive cell platforms similarly benefit from ddPCR-based pharmacokinetic analyses. For TIL products that are not genetically modified, ddPCR assays can be designed around patient-specific T-cell receptor (TCR) sequences identified via next-generation sequencing, enabling longitudinal tracking of individual TCR clonotypes. This approach has shown that clinical responses may correlate more strongly with the persistence of particular TCR clones than with total TIL counts, adding nuance to PK–PD relationships in cell therapy.

Applications of ddCPR in commercial manufacturing and quality control of cell therapies

Within commercial manufacturing, ddPCR provides critical quality control metrics, including transduction efficiency, average vector copy number per cell, and confirmation of the absence of replication-competent lentivirus (RCL). The high precision and reproducibility of ddPCR facilitate compliance with regulatory specifications and support process comparability across manufacturing sites and process changes. Approved CAR-T products now routinely incorporate validated ddPCR assays into release testing.

Post-marketing, ddPCR enables real-world studies examining how variables such as age, disease burden, prior therapies, and concomitant medications influence CAR-T expansion and persistence, informing label refinements, optimization strategies, and patient selection criteria for new indications.

3.  ANTISENSE OLIGONUCLEOTIDES (ASOs)

Unique PK challenges for ASOs

Antisense oligonucleotides (ASOs) constitute a mature yet rapidly evolving therapeutic modality with distinctive pharmacokinetic properties. These synthetic nucleic acids, typically 15–25 nucleotides in length, modulate gene expression via mechanisms such as RNase H–mediated mRNA degradation, splicing modulation, or translational inhibition. ASOs exhibit complex absorption, distribution, metabolism, and excretion (ADME) behavior, including broad tissue distribution, extensive protein binding, and long terminal half-lives in tissues (spanning weeks to months). Such properties demand analytical approaches that can sensitively and specifically quantify intact ASO and, when necessary, distinguish it from metabolites.

Applications of ddPCR in preclinical pharmacokinetic studies

In rodent and NHP toxicology studies, ddPCR offers substantial advantages over hybridization-based ligand-binding assays or HPLC–MS/MS for ASO quantification, particularly for phosphorothioate-modified ASOs that display extensive protein binding and complex matrix interactions. By targeting the unique ASO sequence, ddPCR provides sequence-level specificity that can differentiate intact ASO from closely related degradation products, addressing a key limitation of analytical methods that respond to both parent drug and metabolites.

Tissue distribution studies are especially important for ASOs, which typically distribute broadly after systemic administration but accumulate preferentially in organs such as liver and kidney. ddPCR enables quantification of ASO levels across numerous tissues at low nanogram-per-gram equivalents, supporting detailed biodistribution modeling and safety assessments.

Splice-switching ASOs and pharmacodynamic integration

For splice-switching ASOs such as eteplirsen (Exondys 51) for Duchenne muscular dystrophy, ddPCR plays dual roles in PK and pharmacodynamics (PD). In addition to measuring ASO levels, ddPCR assays can quantify correctly spliced mRNA transcripts (e.g., exon-skipped isoforms), providing an integrated PK–PD readout linking ASO exposure to molecular effect. Multiplex ddPCR assays that simultaneously measure ASO concentration and exon-skipped transcript levels in muscle biopsies have proven especially informative for dose optimization and for establishing mechanistic proof of concept.

Limitations of ddPCR for ASO and siRNA quantification

Despite its strengths in nucleic acid quantification, droplet digital PCR (ddPCR) has important limitations when applied to antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). These therapeutics are short, chemically modified oligonucleotides that do not inherently function as PCR templates in the same manner as genomic DNA or complementary DNA. As a result, ddPCR cannot directly quantify intact ASO or siRNA molecules without additional assay-specific conversion steps.

PCR-based quantification of ASOs and siRNAs typically requires specialized chemistries, such as adapter ligation, primer extension, or other proprietary sample-processing approaches, to render the oligonucleotide amplifiable. The efficiency and reliability of these workflows are highly dependent on oligonucleotide length, sequence, chemical modifications, and the presence of metabolites, introducing additional sources of variability and limiting cross-study comparability.

Furthermore, ddPCR does not inherently distinguish intact parent oligonucleotides from closely related metabolic degradation products. Many ASO and siRNA metabolites retain internal sequence regions recognized by primers and probes, leading to potential overestimation of parent drug concentrations unless assays are carefully engineered to exclude truncated species.

Consequently, while ddPCR can be useful in selected experimental contexts, particularly for mechanistic studies or relative comparisons, it should not be regarded as a universal or superior replacement for established analytical platforms such as LC–MS/MS or hybridization-based assays when precise quantification of intact ASO or siRNA and metabolite resolution are required.

4.  GENE EDITING THERAPEUTICS

CRISPR and Base Editing Technologies

Gene-editing therapeutics employing CRISPR–Cas9, base editors, or prime editors represent the cutting edge of genomic medicine. Unlike conventional gene addition approaches, these platforms introduce permanent sequence modifications at specific genomic loci. PK characterization therefore spans multiple domains: the distribution and persistence of the delivery vehicle (e.g., AAV, lipid nanoparticles, electroporation), the kinetics of editing machinery components (mRNA, protein, or RNP complexes), and, most critically, the magnitude and durability of editing at target and off-target sites.

Applications of ddPCR in preclinical gene editing pharmacokinetics

In mouse and NHP models of in vivo gene editing, ddPCR enables precise and quantitative assessment of editing efficiency across tissues and over time. For AAV-delivered CRISPR therapeutics, multiplex ddPCR assays can simultaneously quantify vector genome copies, Cas9 or editor transgene expression (via RT-ddPCR), and editing outcomes at the desired genomic locus. This integrated analysis reveals temporal relationships between vector exposure, editor expression, and the accumulation and stability of edited alleles.

Assay design for editing quantification requires careful consideration. For non-homologous end joining (NHEJ)–mediated knockouts, assays often exploit the loss of restriction enzyme sites or the presence of characteristic indels. Multiplex ddPCR assays targeting both edited and wild-type alleles allow direct calculation of editing percentages. For homology-directed repair (HDR) or base-editing approaches, allele-specific probes distinguish edited from unedited sequences with single-nucleotide resolution, enabling accurate quantification of precise base changes.

Off-target editing remains a central safety concern. While next-generation sequencing is used to discover potential off-target sites, ddPCR offers higher quantitative accuracy and throughput for longitudinal monitoring of known off-target loci across multiple tissues and timepoints, particularly in long-term toxicology studies. ddPCR-based tracking can detect subtle changes in off-target editing frequencies that may signal clonal expansion or selection.

Applications of ddPCR in ex vivo editing for cell therapies

For ex vivo gene-editing approaches, such as CRISPR-edited hematopoietic stem cells (HSCs) or edited CAR-T cells, ddPCR is used both in manufacturing and in post-infusion monitoring. During production, ddPCR quantifies editing efficiency in the drug product and may serve as a release criterion. Post-infusion, ddPCR enables longitudinal assessment of edited cell persistence and editing stability in different blood lineages and tissues.

Clinical Development

In first-in-human studies of in vivo gene editors, demonstrating editing at the target tissue can be challenging when the tissue is difficult to sample repeatedly. For liver-targeted editors treating conditions such as transthyretin amyloidosis or familial hypercholesterolemia, liver biopsies provide definitive evidence of editing, but their invasiveness limits frequency. ddPCR analysis of circulating cell-free DNA (cfDNA) is being explored as a less invasive surrogate for tissue editing, although correlations with tissue-level editing require careful validation.

For hematologic gene editing targeting HSCs, peripheral blood provides a more accessible compartment. ddPCR quantification of editing frequencies in different leukocyte subsets over time can distinguish editing confined to short-lived progenitors from editing that has successfully engrafted in long-term repopulating stem cells—a key determinant of therapeutic durability and long-term benefit.

Exposure–response modeling in gene-editing trials differs from conventional PK in that the relevant “exposure” metric is editing percentage rather than drug concentration. ddPCR-based editing measurements provide the granular data necessary to correlate editing levels with clinical outcomes, informing whether suboptimal responses reflect inadequate editing (suggesting dose escalation or improved delivery) or editing-independent biological factors.

Safety Monitoring and Long-Term Follow-Up

Given the potential for delayed genotoxicity, gene-editing trials are subject to extended follow-up, often extending 15 years beyond treatment. ddPCR contributes to this long-term safety surveillance by enabling sensitive detection of clonal expansions or lineage-restricted editing patterns that may indicate emerging malignancies or other adverse outcomes. Serial measurement of editing frequencies at multiple loci over years helps build the safety database required for broad clinical adoption of gene-editing therapeutics.

5.  RNA THERAPEUTICS

mRNA and saRNA Pharmacokinetics

The success of mRNA vaccines against COVID-19 has accelerated development of mRNA therapeutics for a broad range of indications, including rare genetic diseases, cancer immunotherapy, and protein replacement. Self-amplifying RNA (saRNA) platforms further extend this paradigm by enabling prolonged protein expression from lower initial doses. The pharmacokinetics of mRNA therapeutics encompass delivery, cellular uptake, endosomal escape, translation, and RNA degradation, all influenced by formulation and delivery route.

Applications of ddPCR on preclinical PK characterization of RNA-based drugs

In rodent and NHP biodistribution studies, ddPCR (typically RT-ddPCR) is used to quantify intact therapeutic mRNA across tissues and time. ddPCR assays targeting the encoded transgene sequence can discriminate exogenous mRNA from endogenous transcripts, enabling detailed mapping of tissue distribution and clearance. Studies of LNP-formulated mRNA vaccines and therapeutics have shown predominant accumulation in liver and spleen after intravenous dosing, with distribution modifiable through LNP composition and route of administration.

Distinguishing intact, translatable mRNA from partially degraded RNA fragments is a particular analytical challenge. Long-amplicon RT-ddPCR assays, or multiplex assays probing both 5′ and 3′ regions, can help identify fragmentation patterns by revealing discrepancies between different assay readouts. For saRNA platforms, ddPCR can separately quantify input RNA and amplified replicon-derived RNA, thereby clarifying whether suboptimal protein expression results from limited delivery, insufficient amplification, or rapid RNA degradation.

Applications of ddPCR in RNA therapeutics clinical trials

In Phase 1 dose-escalation studies, RT-ddPCR is applied to plasma samples to characterize mRNA dose proportionality and clearance profiles, although mRNA concentrations are typically low and short-lived, with terminal half-lives often under 24 hours. For locally administered mRNAs, such as intratumoral cancer immunotherapy or intramuscular protein-replacement candidates, plasma PK may be limited, and biopsy-based tissue PK becomes more informative where feasible.

The encapsulation of mRNA within LNPs adds additional complexity. ddPCR workflows must incorporate steps that disrupt LNPs to release RNA; incomplete disruption leads to underestimation of total mRNA. Conversely, comparing mRNA levels with and without LNP disruption can inform encapsulation efficiency during formulation development.   Immunogenicity-related factors, such as anti-PEG antibodies directed against LNP components, can alter mRNA PK through accelerated clearance or hypersensitivity reactions, and ddPCR-based PK data have been instrumental in identifying such phenomena.

Regulatory Validation and Commercial Viability

Validated RT-ddPCR methods for RNA therapeutics must contend with RNA instability, variable reverse transcription efficiency, and potential interference from abundant endogenous RNAs. Nevertheless, regulatory submissions for several approved RNA therapeutics have included digital PCR–based assays for quantifying drug levels and pharmacodynamic biomarkers, reflecting growing acceptance of these platforms.   As the RNA therapeutic pipeline expands to encompass circular RNAs, longer-acting mRNAs, and combination regimens, ddPCR methodologies continue to evolve in step with emerging analytical needs.

CONCLUSION

Droplet digital PCR has established itself as an indispensable technology for pharmacokinetic characterization of advanced therapeutics across the drug development continuum. Its unique combination of absolute quantification, exceptional analytical sensitivity, robustness in complex matrices, and broad dynamic range addresses many of the analytical challenges posed by viral vectors, cell and gene therapies, antisense oligonucleotides, gene editors, and RNA therapeutics, modalities that increasingly define the future of medicine.

From preclinical toxicology studies that establish first-in-human doses, through pivotal clinical trials that underpin regulatory approval, and onward into commercial manufacturing and post-marketing surveillance, ddPCR provides a quantitative foundation for understanding how these complex therapeutics behave in vivo. Crucially, ddPCR can quantify not only the therapeutic constructs themselves but also their molecular consequences, such as editing outcomes, transgene expression, and target modulation, helping to bridge classical PK and pharmacodynamic assessments.

As therapeutic modalities grow more sophisticated—incorporating combination regimens, targeted delivery systems, and patient-specific customization—analytical demands will intensify in parallel. Ongoing ddPCR innovations, including expanded multiplexing, enhanced throughput, single-cell partitioning, and deeper integration with automation and computational analysis, position the technology to meet these evolving requirements.  Over the coming decade, ddPCR is likely to become as routine for advanced therapeutic PK as LC–MS/MS is for small molecules today, cementing its role as a cornerstone of precision medicine development.

FOR FURTHER READING

General ddPCR principles and PK applications

  1. Hindson BJ et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copies. Anal Chem. 2011;83(22):8604–8610.    https://pubs.acs.org/doi/10.1021/ac202028g
  2. Chan C-H et al. Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis.Sci Rep 2018 Feb 21;8:3432. doi: 10.1038/s41598-018-21041-5
  3. Pinheiro LB et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012;84(2):1003–1011. https://pubs.acs.org/doi/10.1021/ac202578x
  4. Olmedillas-López S et sl. Current and Emerging Applications of Droplet Digital PCR in Oncology: An Updated Review. Mol Diagn Ther. 2022  26:61-87. doi: 10.1007/s40291-021-00562-2.
  5. Long s. In pursuit of sensitivity: Lessons learned from viral nucleic acid detection and quantification on the Raindance ddPCR platform.Methods. 2021 Apr 9;201:82–95. doi: 10.1016/j.ymeth.2021.04.008
  6. Rice N et al. Development of a novel ddPCR assay for nonclinical and clinical pharmacokinetic characterization of ICVB-1042. J Clin Oncol41, e14693(2023) DOI: 10.1200/JCO.2023.41.16_suppl.e14693

 

ddPCR applications in viral vector–based gene therapy

  1. Kavita U et al. PK/PD and Bioanalytical Considerations of AAV-Based Gene Therapies: an IQ Consortium Industry Position Paper. AAPS J 2023. 25(5):78. doi: 10.1208/s12248-023-00842-1.
  2. Shmidt AA, Egorova TV PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations Pharmaceuticals (Basel). 2021 Dec 24;15(1):23. doi: 10.3390/ph15010023
  3. Bosworth A et al. Accurate quantification and characterization of adeno-associated viral vectors. Front Microbiol. 2019;10:1570. doi:10.3389/fmicb.2019.01570.
  4. Clark JR et al. Quantification of adeno-associated viral genomes in purified vector samples by digital droplet polymerase chain reaction. J Vis Exp. 2025;(201):e67252. doi:10.3791/67252.
  5. Powers TW et al. Implementing a robust platform analytical procedure for measuring AAV vector genome concentration. Mol Ther Methods Clin Dev. 2024;32:101381. DOI: 10.1016/j.omtm.2024.101381 ​
  6. Kiladjian A. A Highly Precise Method for the Quantitation of rAAV Cellular Uptake by ddPCR.Hum Gene Ther . 2025 Jul;36(13-14):1004-1011. doi: 10.1089/hum.2025.023. DOI: 10.1089/hum.2025.023
  7. Prantner A, Maar D. Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR. PLoS One. 2023 Jan 25;18(1):e0280242. doi: 10.1371/journal.pone.0280242.
  8. Longacre B ewt al. A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears. J Vis Exp. 2023 Jul 14;(197). doi: 10.3791/65495.
  9. Blay E et al. PCR-based analytics of gene therapies using adeno-associated virus vectors: Considerations for cGMP method development. Molecular Therapy Methods & Clinical Development, 31:101132. DOI: 10.1016/j.omtm.2023.101132 .
  10. Green EA, Lee KH. Analytical methods to characterize recombinant adeno-associated virus vectors and the benefit of standardization and reference materials. Curr Opin Biotechnol. 2021 Oct;71:65-76. doi: 10.1016/j.copbio.2021.06.025.

 

Cell and gene therapies  

  1. Marton C et al. Harmonisation of quality control tests for academic production of CAR-T cells: a position paper from the WP-bioproduction of the UNITC consortium. Bone Marrow Transplant. 2025 Sep;60(9):1209-1217. doi: 10.1038/s41409-025-02637-8. Epub 2025 May 29. Erratum in: Bone Marrow Transplant. 2025 Sep;60(9):1300. doi: 10.1038/s41409-025-02672-5.
  2. Ma J  er al. An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products. Immunooncol Technol. 2024 Dec 4;25:101031. doi: 10.1016/j.iotech.2024.101031. PMID: 40236328; PMCID: PMC11997555.
  3. Iida T et al . Accurate vector copy number determination in gammaretroviral vector producer cell clones using triplex digital droplet PCR. J Virol Methods. 2025 Feb;332:115075. doi: 10.1016/j.jviromet.2024.115075.
  4. Wiedemann G  et al. A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications. Int J Mol Sci. 2024 Aug 6;25(16):8556. doi: 10.3390/ijms25168556.
  5. Galli E et al. Unlocking Predictive Power: Quantitative Assessment of CAR-T Expansion with Digital Droplet Polymerase Chain Reaction (ddPCR). Int J Mol Sci. 2024 Feb 26;25(5):2673. doi: 10.3390/ijms25052673
  6. Kandell J et al. Universal ddPCR-based assay for the determination of lentivirus infectious titer and lenti-modified cell vector copy number. Mol Ther Methods Clin Dev. 2023 Sep 23;31:101120. doi: 10.1016/j.omtm.2023.101120.

 

Antisense oligonucleotides (ASOs)

  1. Verheul RC et al. Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy. PLoS One. 2016 Sep 9;11(9):e0162467. doi: 10.1371/journal.pone.0162467.
  2. Mitake M et al. Imprinting analysis by droplet digital PCR coupled with locked nucleic acid TaqMan probes. Epigenetics. 2021 Jun-Jul;16(7):729-740. doi: 10.1080/15592294.2020.1823160.
  3. Hiller M et al. Exon 51 Skipping Quantification by Digital Droplet PCR in del52hDMD/mdx Mice. Methods Mol Biol. 2018;1828:249-262. doi: 10.1007/978-1-4939-8651-4_15.

 

Gene editing therapeutics

  1. Wang K et al. A Novel Quantification Method for Gene-Edited Animal Detection Based on ddPCR. Biology (Basel). 2025 Feb 14;14(2):203. doi: 10.3390/biology14020203.
  2. Nilsri N et al. CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents. Int J Mol Sci. 2025 May 11;26(10):4600. doi: 10.3390/ijms26104600.
  3. Rose, J et al. Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics. Nat Methods 14, 891–896 (2017). https://doi.org/10.1038/nmeth.4368

 

Image Note: The image in this news article is a depiction of the concept visualized by Google Gemini.

Disclaimer: This blog post is intended solely for educational and scientific informational purposes. Any mention of therapeutic drug names, including FDA-approved medications, is for the purpose of accurate reporting and discussion of biomedical research and does not constitute medical advice, endorsement, or promotion. Readers should not interpret the content as a recommendation for any specific treatment. Always consult a qualified healthcare professional for medical advice or treatment decisions.

 

About Marin Biologic Laboratories

Our Recent Publication/Meeting Presentation on Gene Therapy

1. Development of VNX-101, an Adeno-Associated Virus with Less Immunogenicity and Efficient Long-Term Expression of a CD19 T-Cell Engager. Molecular Therapy Methods & Clinical Development, published online July 24, 2025.

3. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. Journal of Immunological Methods. 2025. 545-114004.

3. Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217.

4. American Society of Hematology (ASH) Annual Meeting 2024.
Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

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Watch the following video and explore our latest presentation on the development and validation of potency and pharmacokinetic (PK) assays for AAV vectors, highlighting innovative methodologies and industry-leading expertise.

 

 

Download the full presentation: Development of Custom Cell Based and In vitro Potency and Pharmacokinetics (PK) Assays for AAV vectors- Marin biologic Laboratories

 

Development of Cell-Based Potency Assays: Case Studies and Blogs from Marin Biologic Laboratories (MarinBio)

 

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