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Comparison of RT-qPCR With Branched DNA to Quantify a Lipid Nanoparticle-Encapsulated mRNA Therapeutic in Serum and Liver Tissue Samples From Nonclinical PK Studies

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AAPS JOURNAL 2025年第1期27卷 1-10页

作者： Ortiz, Jessica Brunner, Laura Ci, Lei Baek, Rena Jani, Darshana marshall, jean-claude Pennucci, Jason Moderna Inc Cambridge MA 02142 USA

While the branched DNA (bDNA) assay is an established bioanalytical method for measurement of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) pharmacokinetic parameters, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has been considered as an alternative platform. RT-qPCR and bDNA platforms were compared for sensitivity, specificity, correlation, and overall assay performance using serum and tissue samples from 2 nonclinical mouse studies of a therapeutic mRNA candidate, LNP-PAH-mRNA, which encodes for human phenylalanine hydroxylase enzyme. Pharmacokinetic parameter noncompartmental analysis was completed using Phoenix WinNonlin. The assays were compared using simple linear regression and Bland-Altman analyses. Sensitivity ranged from 0.05 to 6.40 ng/mL for the bDNA assays, from 0.00000761 to 7.61 ng/mL in serum, and from 0.000179 to 179 ng/g in tissue for the RT-qPCR assay. Inter-assay accuracy was within +/- 10%, inter-assay precision was <= 10%, and the total error for both assays was <= 20%. RT-qPCR serum mRNA concentrations were 2- to fourfold lower compared with the bDNA assay, whereas tissue samples were comparable between assays. A linear relationship with - 0.37 to - 0.02 systematic bias demonstrated acceptable concordance. Bland-Altman plots demonstrated close equivalence, with a negative bias of < 0.5, and >= 95% of the data points were within the 95% limits of agreement. The comparison of the RT-qPCR with bDNA assay platforms for quantification of pharmacokinetic properties of an mRNA-LNP therapeutic has demonstrated acceptable concordance. This comparison reinforces the use of the RT-qPCR, a widely accessible strategy, as an alternative platform for the quantification of subsequent mRNA-LNP therapeutics.

关键词： bDNA bioanalytical mRNA therapeutics pharmacokinetics RT-qPCR

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Protein-protein interactions: a mechanism regulating the anti-metastatic properties of Nm23-H1

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NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY 2011年第4-5期384卷 351-362页

作者： Marino, Natascia marshall, jean-claude Steeg, Patricia S. NIH Bethesda MD 20892 USA NCI Womens Canc Sect Mol Pharmacol Lab Ctr Canc Res Bethesda MD 20892 USA

Nm23-H1, also known as NDPK-A, was the first of a class of metastasis suppressor genes to be identified. Overexpression of Nm23-H1 in metastatic cell lines (melanoma, breast carcinoma, prostate, colon, hepatocellular, and oral squamous cell carcinoma) reduced cell motility in in vitro assays and metastatic potential in xenograft models, without a significant effect on primary tumor size. The mechanism of Nm23-H1 suppression of metastasis, however, is incompletely understood. Nm23-H1 has been reported to bind proteins, including those in small G-protein complexes, transcriptional complexes, the Map kinase, the TGF-beta signaling pathways and the cytoskeleton. Evidence supporting these associations is presented together with evidence of resultant biochemical and phenotypic consequences of association. Cumulatively, the data suggest that part of the anti-metastatic function of Nm23-H1 lies in pathways that it interrupts via binding and inactivation of proteins.

关键词： Nm23-H1 Protein interactions Metastasis

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The Nm23-H1 metastasis suppressor as a translational target

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EUROPEAN JOURNAL OF CANCER 2010年第7期46卷 1278-1282页

作者： marshall, jean-claude Collins, Joshua Marino, Natascia Steeg, Patricia NCI Womens Canc Sect Mol Pharmacol Lab Ctr Canc Res Bethesda MD 20892 USA

Nm23 was the first of what has become a field of over 20 known metastasis suppressor genes (MSGs). Since the discovery of Nm23 in 1988, a variety of mechanisms have been attributed to its activity, including a histidine kinase activity, binding of other proteins to regulate metastatic formation, and altered gene expression downstream of Nm23. Here, we will review current efforts to translate the previous work done on this MSG into the clinic, including high-dose medroxyprogesterone acetate (MPA), which has been shown to upregulate Nm23 expression. In addition, we will detail a new potential target downstream of Nm23. LPA1 is one of a group of known cell surface receptors for lysophosphatidic acid (LPA), which has been shown to be inversely correlated with Nm23 expression. A specific LPA1 antagonist could conceivably mimic the effects of Nm23 by downregulating the activity of the LPA1 pathway, which would be of considerable interest for potential clinical use. Published by Elsevier Ltd.

关键词： Nm23-H1 Metastasis

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Clinical-translational strategies for the elevation of Nm23-H1 metastasis suppressor gene expression

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MOLECULAR AND CELLULAR BIOCHEMISTRY 2009年第1-2期329卷 115-120页

作者： marshall, jean-claude Lee, Jong Heun Steeg, Patricia S. NCI Womens Canc Sect Mol Pharmacol Lab Ctr Canc Res Bethesda MD 20892 USA

Interruption of the tumor metastatic process is a new, thought provoking molecular target for the treatment of cancer. The Nm23-H1 metastasis suppressor gene stands as a validated molecular target owing to its reduced expression in many aggressive human tumors, and the reduction in metastatic potential in vivo upon re-expression in multiple cell lines. Several compounds have been identified which elevate Nm23-H1 expression in vitro including indomethacin, gamma Linolenic Acid, trichostatin A, 5-aza-deoxycytidine, and high dose medroxyprogesterone acetate. Using a model of lung metastatic colonization by MDA-MB-231 human breast carcinoma cells, we demonstrated that high dose MPA reduced the formation of overt lung metastases by 37-46% and those metastases that formed were statistically smaller. A Phase II clinical trial of high dose MPA, alone or in combination with metronomic chemotherapy has recently opened.

关键词： Nm23 Metastasis suppressor genes Gene expression

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Bioanalysis of adeno-associated virus gene therapy therapeutics: regulatory expectations

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BIOANALYSIS 2019年第21期11卷 2011-2024页

作者： Gorovits, Boris marshall, jean-claude Smith, Justin Whiteley, Laurence O. Neubert, Hendrik Pfizer Inc Biomed Design 1 Burtt Rd Andover MA 01810 USA Pfizer Inc Early Clin Dev Eastern Point Rd Groton CT 06340 USA Pfizer Inc Drug Safety Res & Dev 1 Portland St Cambridge MA 02139 USA

The number of gene therapy (GTx) modality therapies in development has grown significantly in the last few years. Adeno-associated virus (AAV)-based delivery approach has become most prevalent among other virus-based GTx vectors. Several regulatory guidelines provide the industry with general considerations related to AAV GTx development including discussion and recommendations related to highly diverse bioanalytical support of the AAV-based therapeutics. This includes assessment of pre- and post-treatment immunity, evaluation of post-treatment viral shedding and infectivity, as well as detection of transgene protein expression. An overview of the current regulatory recommendations as found in currently active and published draft US FDA and EMA guidance or guideline documents is presented herein.

关键词： gene therapy genome integration immune response infectivity neutralizing antibody total antibody transgene protein viral vector shedding

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Altered gene and protein expression by Nm23-H1 in metastasis suppression

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MOLECULAR AND CELLULAR BIOCHEMISTRY 2009年第1-2期329卷 141-148页

作者： Lee, Jong Heun marshall, jean-claude Steeg, Patricia S. Horak, Christine E. NCI Womens Canc Sect Mol Pharmacol Lab Ctr Canc Res Bethesda MD 20892 USA

Metastasis suppressor genes (MSG) are characterized by their ability to inhibit the formation of metastasis, while not affecting the growth of the primary tumor in vivo. Nm23-H1, the first MSG to be characterized, has been shown to alter both gene and protein expression in cancer cells. Recently, microarray expression profiling revealed that Nm23-H1 downregulated EDG2, which encodes for a lysophosphatidic acid (LPA) receptor. Reintroduction of EDG2 into cells that express Nm23-H1 overcame the metastasis suppressive ability of Nm23-H1 in both in vivo pulmonary colonization and spontaneous metastasis assays. In addition, isotope capture affinity tag (ICAT) proteomic analysis was performed to identify differentially expressed proteins not accounted for by microarray analysis. ICAT identified several differentially regulated proteins, including GEMIN5, a protein involved in differential mRNA splicing. The contribution of alternative mRNA splicing to cancer and cancer metastasis is poorly defined. It is possible that Nm23-H1, through the regulation of RNA processing proteins, may play a role in proteome stability.

关键词： Nm23 Metastasis suppressor genes Gene expression

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Assessment of inter-racial variability in CYP3A4 activity and inducibility among healthy adult males of Caucasian and South Asian ancestries

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EUROPEAN JOURNAL OF CLINICAL PHARMACOLOGY 2018年第7期74卷 913-920页

作者： van Dyk, Madele marshall, jean-claude Sorich, Michael J. Wood, Linda S. Rowland, Andrew Flinders Univ S Australia Coll Med & Publ Hlth Adelaide SA 5042 Australia Pfizer Worldwide Res & Dev Precis Med Groton CT USA

Cytochrome P450 (CYP) 3A4 is responsible for the metabolism of more than 30% of clinically used drugs. Inherent between subject variability in clearance of CYP3A4 substrates is substantial;by way of example, midazolam clearance varies by > 10-fold between individuals before considering the impact of extrinsic factors. Relatively little is known about inter-racial variability in the activity of this enzyme. This study assessed inter-racial variability in midazolam exposure in a cohort (n = 30) of CYP3A genotyped, age-matched healthy males of Caucasian and South Asian ancestries. Midazolam exposure was assessed at baseline, following 7 days of rifampicin and following 3 days of clarithromycin. The geometric mean baseline midazolam area under the plasma concentration curve (AUC(0-6)) in Caucasians (1057 mu g/L/min) was 27% greater than South Asians (768 mu g/L/min). Similarly, the post-induction midazolam AUC(0-6) in Caucasians (308 mu g/L/min) was 50% greater than South Asians (154 mu g/L/min), while the post-inhibition midazolam AUC(0-6) in Caucasians (1834 mu g/L/min) was 41% greater than South Asians (1079 mu g/L/min). The difference in baseline AUC(0-6) between Caucasians and South Asians was statistically significant (p 0.05), and a trend toward significance (p = 0.067) was observed for the post-induction AUC(0-6) ratio, in both unadjusted and genotype adjusted analyses. Significantly higher midazolam clearance was observed in healthy age-matched males of South Asian compared to Caucasian ancestry that was not explained by differences in the frequency of CYP3A genotypes.

关键词： Caucasian Cytochrome P450 3A4 Inter-racial variability Inducibility Pharmacokinetics South Asian

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Current challenges and opportunities for pharmacogenomics: perspective of the Industry Pharmacogenomics Working Group (I-PWG)

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HUMAN GENETICS 2022年第6期141卷 1165-1173页

作者： Bienfait, Karina Chhibber, Aparna marshall, jean-claude Armstrong, Martin Cox, Charles Shaw, Peter M. Paulding, Charles Bristol Myers Squibb Princeton NJ 08543 USA Merck & Co Inc Kenilworth NJ USA Pfizer Groton CT 06340 USA UCB Chemin Foriest 1 B-1420 Braine Lalleud Belgium GSK Med Res Ctr Gunnels Wood Rd Stevenage SG1 2NY Herts England Regeneron Pharmaceut Inc Tarrytown NY 10591 USA

Pharmaceutical companies have increasingly utilized genomic data for the selection of drug targets and the development of precision medicine approaches. Most major pharmaceutical companies routinely collect DNA from clinical trial participants and conduct pharmacogenomic (PGx) studies. However, the implementation of PGx studies during clinical development presents a number of challenges. These challenges include adapting to a constantly changing global regulatory environment, challenges in study design and clinical implementation, and the increasing concerns over patient privacy. Advances in the field of genomics are also providing new opportunities for pharmaceutical companies, including the availability of large genomic databases linked to patient health information, the growing use of polygenic risk scores, and the direct sequencing of clinical trial participants. The Industry Pharmacogenomics Working Group (I-PWG) is an association of pharmaceutical companies actively working in the field of pharmacogenomics. This I-PWG perspective will provide an overview of the steps pharmaceutical companies are taking to address each of these challenges, and the approaches being taken to capitalize on emerging scientific opportunities.

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The role of metastasis, suppressor genes in metastatic dormancy

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APMIS 2008年第7-8期116卷 586-601页

作者： Horak, Christine E. Lee, Jong Heun marshall, jean-claude Shreeve, S. Martin Steeg, Patricia S. NCI Ctr Canc Res Mol Pharmacol Lab Womens Canc Sect Bethesda MD 20892 USA

Metastasis suppressor genes (MSGs) are defines by their ability to inhibit overt metastasis in a secondary organ without affecting tumor growth at the primary site. Over 20 MSGs have been confirmes in vivo. This class of genes is only unified by their capacity to supress metastasis, as they encode for proteins with a wide range of biochemical activities that are components of a varity of signaling pathways. In addition, metastasis supressors impinge upon different stages of the metastatic cascade to manifest their supressive effects. The MSGs KISSI, KAII, MK K4/7 and Nm23-HI promote tumor dormancy at the metastatic site, since tumor cells with induced expression of these MSGs disseminate, but do not form overt metastases in the secondary organ throughout the duration of a metastasis assay. Evidence suggests of solitary cells at the secondary site. KAII induces growth arrest prior to extravasation by binding a vascular endothelial cell surface marker. MK K4, MKK7 and Nm23-HI appear to promote dormancy of micrometastatic colonies, after disseminated tumor cells have undergone several rounds of proliferation. Other MSGs may also function in tumor dormancy, but so far their role has not been fully elucidated. Therapeutic approaches that either mimic the effects of MSGs or re-establish MSg expression in metastatic lesions may hold promise fo the establishment or maintenance of dormancy.

关键词： dormancy KAII KISSI metastasis metastasis supressor gene MKK4 MKK7 Nm23

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Transcriptional profiling of human uveal melanoma from cell lines to intraocular tumors to metastasis

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CLINICAL & EXPERIMENTAL METASTASIS 2007年第5期24卷 353-362页

作者： marshall, jean-claude Nantel, Andre Blanco, Paula Ash, Josee Cruess, Stephanie R. Burnier, Miguel N., Jr. McGill Univ Henry C Witelson Ocular Pathol Lab & Registry Montreal PQ H3A 2B4 Canada Natl Res Council Canada Biotechnol Res Inst Montreal PQ H4P 2R2 Canada

Uveal melanoma is the most common primary intraocular tumor in adults and exclusively disseminates haematogenously in order to form metastases. The aim of this study was to measure the transcriptional profiles of human uveal melanoma cells isolated from a primary intraocular tumor, circulating malignant cells (CMCs), and metastases in order to elucidate the changes in gene expression associated with this progression. Human EST microarrays and universal reference RNA were used to measure the differences between tissue samples isolated from an immunosuppressed xenograft rabbit model of uveal melanoma. Cells were isolated from a single rabbit at the time of sacrifice from an intraocular tumor, peripheral blood, and metastasis. RNA was extracted from each sample and subjected to transcriptional profiling analysis. Results were compared to the transcriptional profiles previously obtained from the original cell line used for intraocular injections. Changes were verified using real-time PCR analysis. A total of 314 significant changes in gene expression were seen from the intraocular tumor to metastasis, as determined by transcript abundance. Principle Components Analysis was used to cluster these changes into four distinct groups. An additional 61 statistically significant changes were observed between the recultured and CMCs, with the latter believed to represent an intermediate step in the progression from intraocular tumor to metastasis. In conclusion, we have produced a detailed analysis of the transcriptional changes that take place as human uveal melanoma cells evolve from a primary tumor to metastasis in a xenograft animal model, including the decrease in expression of specific melanoma markers.

关键词： uveal melanoma circulating malignant cells metastasis animal model

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