The emergence of nucleic acid (NA) therapeutics, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), which are usually delivered directly, and messenger RNAs (mRNAs), which are typically encapsulated in lipid nanoparticles (LNPs), marks a transformative era in precision medicine. While these therapies offer precise approaches for gene regulation or expression, they can trigger unwanted innate and/or adaptive immune responses that can either have no significant impact or adversely affect treatment efficacy and/or patient safety. Consequently, therapies where an adaptive immune response is desired, such mRNA/LNP-based vaccines against infectious diseases or cancer are out of scope of this article. In the present work, the Innovation and Quality Consortium Nucleic Acids Immunogenicity Working Group examines how the various components of NA-based therapies might contribute to their immunogenic potential and describes risk mitigation strategies through product design adaptations during early development stages. In addition, a comprehensive immunogenicity risk assessment framework is described, allowing to effectively define a tailored clinical testing strategy for different NA modalities with varying immunogenicity (IG) consequences. A streamlined monitoring strategy is recommended when minimal impact is expected, whereas extensive testing is suggested when safety concerns arise. Overall, these recommendations ensure that safe and effective NA-based therapies reach patients with an appropriate assessment of the IG potential.

Adeno-associated virus (AAV)-based gene therapies are emerging as transformative treatments for serious diseases; however, determining the optimal duration of nonclinical toxicity studies remains a key regulatory and scientific question. To address this, the EFPIA Gene Therapy Working Group surveyed 24 AAV gene therapy programs across 13 companies to assess current practices and the value of long-term (≥6 months) toxicity studies. Results showed that ≤3-month studies were sufficient to characterize the toxicology profile in 87.5% of programs that completed a toxicity assessment in a ≥6-month long-term chronic studies, with only one program identifying new toxicities in longer chronic studies with impact on clinical development. Common AAV-related toxicities, such as liver and dorsal root ganglia effects, were observed within the first 6 weeks post-administration. Longer studies were often driven by sponsor's perception based on internal experience or need to assess durability, rather than regulatory requirements. These findings aligned with regulatory reviews of approved AAV products (e.g., Zolgensma, Luxturna, Roctavian) that consistently demonstrated the adequacy of ≤3-month studies for approved and marketed products. The outcome of this survey supports a risk-based, science-driven approach to in vivo study duration, emphasizing that shorter-term studies are generally sufficient for identifying relevant toxicities associated with AAV-based gene therapies. Embracing this approach can reduce animal use, accelerate development timelines, and support harmonized regulatory expectations for AAV gene therapy products.

Gene therapy medicinal products (GTMPs) are currently undergoing intense industrial expansion and technological advancement. However, one issue facing development of most GTMPs is the generation of unwanted immune responses. Immunomodulatory strategies are also often applied in conjunction with GTMP administration to suppress or enhance these responses. This review focusses on the global regulatory requirements for immunogenicity assessments and immunomodulation in relation to GTMPs. The specific aims are to (1) identify the principal international guidelines; (2) identify areas of concordance and discrepancy between guidelines; (3) propose areas where guidelines could be harmonized; and (4) predict areas, which future guidance may address. Methodologies used included surveillance of literature, international guidelines, advocacy initiatives, and compilation of previous regulatory advice received. Overall, there is a clear absence of and need for GTMP-specific guidance on immunogenicity and immunomodulation. Several specific measures and areas for future regulatory harmonization and coverage are proposed.

Recombinant adeno-associated virus (AAV) vectors are the leading delivery vehicle used for in vivo gene therapies. AntiAAV antibodies (AAV Abs) can interact with the viral capsid component of an AAV-based gene therapy (GT). Therefore, patients with preexisting AAV Abs (seropositive patients) are often excluded from GT trials to prevent treatment of patients who are unlikely to bene fi t s or may have a higher risk for adverse events outweighing treatment bene fi ts. On the contrary, unnecessary exclusion of patients with high unmet medical need should be avoided. Instead, a risk -bene fi t assessment that weighs the potential risks due to seropositivity vs. severity of disease and available treatment options, should drive the decision if patient selection is required. Assays for patient selection must be validated according to their intended use following national regulations/standards for diagnostic assays in appropriate laboratories. In this review, we summarize the current process of patient selection, including assay cutoff criteria and related assay validation approaches. We further provide considerations on regulatory requirements for the development of in vitro diagnostic tests supporting market authorization of a corresponding GT.

Chapter 4 Nonclinical and Clinical Study Considerations for Biodistribution, Shedding, and Pharmacokinetics/Pharmacodynamics Manuela Braun, Manuela Braun 1 Preclinical Development, Project Management, Bayer AG, Berlin, GermanySearch for more papers by this authorKefeng Sun, Kefeng Sun 2 Quantitative Clinical Pharmacology, Data Sciences Institute, Takeda Development Center Americas, Cambridge, MA, USASearch for more papers by this author Manuela Braun, Manuela Braun 1 Preclinical Development, Project Management, Bayer AG, Berlin, GermanySearch for more papers by this authorKefeng Sun, Kefeng Sun 2 Quantitative Clinical Pharmacology, Data Sciences Institute, Takeda Development Center Americas, Cambridge, MA, USASearch for more papers by this author Book Editor(s):Yanmei Lu, Yanmei Lu Sangamo Therapeutics, Brisbane, California, United StatesSearch for more papers by this authorBoris Gorovits, Boris Gorovits Sana Biotechnology, Cambridge, Massachusetts, United StatesSearch for more papers by this author First published: 16 February 2024 https://doi.org/10.1002/9781119852810.ch4 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onEmailFacebookTwitterLinkedInRedditWechat Summary In nonclinical and clinical studies, the biodistribution, encompassing distribution, persistence, and clearance of a gene therapeutic product within the body as well as the shedding, the release outside the body, should be assessed as appropriate. In this chapter, we discuss the design, execution, and reporting of the biodistribution and shedding assessment during nonclinical and clinical development considering regulatory requirements. We describe the gaps and challenges of these assessments in the different development phases. The pharmacokinetic/pharmacodynamic (PK/PD) analysis for a gene therapy product is intricately linked to its profiles of biodistribution and transgene product exposure. The PK/PD readout and analysis should integrate all available data sources, including biodistribution, as measured by the vector genome, transgene product levels, biomarker response, and efficacy endpoints. Clinical dose selection for AAV is informed by both safety and efficacy data in nonclinical models and human, and should consider both allometric scaling and mechanistic methods. References ICH guideline S12 on nonclinical biodistribution considerations for gene therapy products 2023 . Available from: https://www.fda.gov/media/167605/download . Google Scholar European Medicines Agency ( 2008 ). Guideline on the nonclinical studies required before first clinical use of gene therapy medicinal products. EMEA/CHMP/GTWP/125459/2006 . Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-non-clinical-studies-required-first-clinical-use-gene-therapy-medicinal-products_en.pdf . Google Scholar European Medicines Agency Reflection paper on quality, non-clinical and clinical issues related to the development of recombinant adeno- associated viral vectors. EMEA/CHMP/GTWP/587488/2007 Rev. 1 . Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-quality-non-clinical-clinical-issues-related-development-recombinant-adeno_en.pdf . Google Scholar European Medicines Agency ( 2018 ). Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products. EMA/CAT/80183/2014 . Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-quality-non-clinical-clinical-aspects-gene-therapy-medicinal-products_en.pdf . Google Scholar European Medicines Agency ( 2019 ). Guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products in clinical trials – scientific guideline . Available from: https://www.ema.europa.eu/documents/scientific-guideline/draft-guideline-quality-non-clinical-clinical-requirements-investigational-advanced-therapy_en.pdf . Google Scholar European Medicines Agency ( 2013 ). Guideline on the risk-based approach according to annex I, part IV of Directive 2001/83/EC applied to Advanced therapy medicinal products. EMA/CAT/CPWP/686637/2011 . Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-risk-based-approach-according-annex-i-part-iv-directive-2001/83/ec-applied-advanced-therapy-medicinal-products_en.pdf . Google Scholar U.S. Food and Drug Administration ( 2013 ). Preclinical assessment of investigational cellular and gene therapy products: guidance for industry . Available from: https://www.fda.gov/media/87564/download . Google Scholar U.S. Food and Drug Administration 2020 Long term follow-up after administration of human gene therapy products: guidance for industry . Available from: https://www.fda.gov/media/113768/download . Google Scholar U.S. Food and Drug Administration ( 2015 ). Design and analysis of shedding studies for virus or bacteria-based gene therapy and oncolytic products: guidance for industry . Available from: https://www.fda.gov/files/vaccines%2C%20blood%20%26%20biologics/published/Design-and-Analysis-of-Shedding-Studies-for-Virus-or-Bacteria-Based-Gene-Therapy-and-Oncolytic-Products--Guidance-for-Industry.pdf . Google Scholar International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use considerations: General principles to address virus and vector shedding. EMEA/CHMP/ICH/449035/2009 . ( 2009 ). Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/international-conference-harmonisation-technical-requirements-registration-pharmaceuticals-human-use_en-10.pdf . Google Scholar International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Final Concept Paper, M6: Guideline on Virus and Gene Therapy Vector Shedding and Transmission . ( 2009 ). Available from: https://database.ich.org/sites/default/files/M6_Concept_Paper.pdf . Google Scholar International Pharmaceutical Regulators Programme ( 2018 ). Expectations for Biodistribution (BD) Assessments for Gene Therapy (GT) Products . Available from: https://admin.iprp.global/sites/default/files/2018-09/IPRP_GTWG_ReflectionPaper_BD_Final_2018_0713.pdf . Google Scholar Assaf , B.T. and Whiteley , L.O. ( 2018 ). Considerations for preclinical safety assessment of adeno-associated virus gene therapy products . Toxicol. Pathol. 46 ( 8 ): 1020 – 1027 . 10.1177/0192623318803867 CASPubMedWeb of Science®Google Scholar Korneyenkov , M.A. and Zamyatnin , A.A. Jr. ( 2021 ). Next step in gene delivery: modern approaches and further perspectives of AAV Tropism modification . Pharmaceutics 13 ( 5 ): 750 . 10.3390/pharmaceutics13050750 CASPubMedGoogle Scholar Bolt , M.W. , Whiteley , L.O. , Lynch , J.L. et al. ( 2020 ). Nonclinical studies that support viral vector-delivered gene therapies: an EFPIA gene therapy working group perspective . Mol. Ther. Methods Clin. Dev. 19 : 89 – 98 . 10.1016/j.omtm.2020.08.017 CASPubMedGoogle Scholar Wright , J.F. ( 2014 ). Product-related impurities in clinical-grade recombinant AAV vectors: characterization and risk assessment . Biomedicine 2 ( 1 ): 80 – 97 . Google Scholar Saraiva , J. , Nobre , R.J. , and Pereira de Almeida , L. ( 2016 ). Gene therapy for the CNS using AAVs: the impact of systemic delivery by AAV9 . J. Control. Release 241 : 94 – 109 . 10.1016/j.jconrel.2016.09.011 CASPubMedWeb of Science®Google Scholar Samaranch , L. , Salegio , E.A. , San Sebastian , W. et al. ( 2012 ). Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates . Hum. Gene Ther. 23 ( 4 ): 382 – 389 . 10.1089/hum.2011.200 CASPubMedWeb of Science®Google Scholar Miyake , N. , Miyake , K. , Yamamoto , M. et al. ( 2011 ). Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors . Brain Res. 1389 : 19 – 26 . 10.1016/j.brainres.2011.03.014 CASPubMedGoogle Scholar Schenk-Braat , E.A. , van Mierlo , M.M. , Wagemaker , G. et al. ( 2007 ). An inventory of shedding data from clinical gene therapy trials . J. Gene Med. 9 ( 10 ): 910 – 921 . 10.1002/jgm.1096 PubMedWeb of Science®Google Scholar Gorovits , B. , Azadeh , M. , Buchlis , G. et al. ( 2021 ). Evaluation of the humoral response to adeno-associated virus-based gene therapy modalities using total antibody assays . AAPS J. 23 ( 6 ): 108 . 10.1208/s12248-021-00628-3 CASPubMedWeb of Science®Google Scholar Gorovits , B. , Fiscella , M. , Havert , M. et al. ( 2020 ). Correction to: recommendations for the development of cell-based anti-viral vector neutralizing antibody assays . AAPS J. 22 ( 2 ): 42 . 10.1208/s12248-020-0425-8 PubMedGoogle Scholar Gorovits , B. , Fiscella , M. , Havert , M. et al. ( 2020 ). Recommendations for the development of cell-based anti-viral vector neutralizing antibody assays . AAPS J. 22 ( 2 ): 24 . 10.1208/s12248-019-0403-1 PubMedGoogle Scholar Gorovits , B. , Marshall , J.C. , Smith , J. et al. ( 2019 ). Bioanalysis of adeno-associated virus gene therapy therapeutics: regulatory expectations . Bioanalysis 11 ( 21 ): 2011 – 2024 . 10.4155/bio-2019-0135 CASPubMedGoogle Scholar Ferla , R. , Alliegro , M. , Marteau , J.B. et al. ( 2017 ). Non-clinical safety and efficacy of an AAV2/8 vector administered intravenously for treatment of mucopolysaccharidosis Type VI . Mol. Ther. Methods Clin. Dev. 6 : 143 – 158 . 10.1016/j.omtm.2017.07.004 CASPubMedGoogle Scholar Favaro , P. , Downey , H.D. , Zhou , J.S. et al. ( 2009 ). Host and vector-dependent effects on the risk of germline transmission of AAV vectors . Mol. Ther. 17 ( 6 ): 1022 – 1030 . 10.1038/mt.2009.56 CASPubMedWeb of Science®Google Scholar Nathwani , A.C. , Rosales , C. , McIntosh , J. et al. ( 2011 ). Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins . Mol. Ther. 19 ( 5 ): 876 – 885 . 10.1038/mt.2010.274 CASPubMedWeb of Science®Google Scholar Pasi , K.J. , Rangarajan , S. , Mitchell , N. et al. ( 2020 ). Multiyear Follow-up of AAV5-hFVIII-SQ Gene Therapy for Hemophilia A . N. Engl. J. Med. 382 ( 1 ): 29 – 40 . 10.1056/NEJMoa1908490 CASPubMedWeb of Science®Google Scholar Manno , C.S. , Pierce , G.F. , Arruda , V.R. et al. ( 2006 ). Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response . Nat. Med. 12 ( 3 ): 342 – 347 . 10.1038/nm1358 CASPubMedWeb of Science®Google Scholar Nathwani , A.C. , Reiss , U.M. , Tuddenham , E.G. et al. ( 2014 ). Long-term safety and efficacy of factor IX gene therapy in hemophilia B . N. Engl. J. Med. 371 ( 21 ): 1994 – 2004 . 10.1056/NEJMoa1407309 CASPubMedWeb of Science®Google Scholar Brunetti-Pierri , N. , Ferla , R. , Ginocchio , V.M. et al. ( 2022 ). Liver-directed adeno-associated virus-mediated gene therapy for mucopolysaccharidosis Type VI . NEJM Evidence 1 ( 7 ): https://doi.org/10.1056/EVIDoa2200052 . 10.1056/EVIDoa2200052 PubMedGoogle Scholar Duan , D. and Systemic , A.A.V. ( 2018 ). Micro-dystrophin gene therapy for Duchenne muscular dystrophy . Mol. Ther. 26 ( 10 ): 2337 – 2356 . 10.1016/j.ymthe.2018.07.011 CASPubMedWeb of Science®Google Scholar uniQure Announces Second Quarter 2022 Financial Results and Highlights Recent Company Progress . ( 2022 ). Available from: https://www.globenewswire.com/en/news-release/2022/08/08/2493736/0/en/uniQure-Announces-Second-Quarter-2022-Financial-Results-and-Highlights-Recent-Company-Progress.html . Google Scholar Chand , D.H. , Zaidman , C. , Arya , K. et al. ( 2021 ). Thrombotic microangiopathy following onasemnogene Abeparvovec for spinal muscular atrophy: a case series . J. Pediatr. 231 : 265 – 268 . 10.1016/j.jpeds.2020.11.054 CASPubMedWeb of Science®Google Scholar U.S. Food and Drug Administration (FDA) ( 2021 ). Cellular, Tissue, and Gene Therapies Advisory Committee (CTGTAC) meeting #70, transcript for day 2 open session . Available from: https://www.fda.gov/media/154121/download . Google Scholar U.S. Food and Drug Administration (FDA) ( 2021 ). Cellular, Tissue, and Gene Therapies Advisory Committee (CTGTAC) meeting #70: Toxicity Risks of Adeno-associated Virus (AAV) Vectors for Gene Therapy (GT), briefing document . Available from: https://www.fda.gov/media/151599/download . Google Scholar Watkins , P.B. ( 2021 ). Liver injury due to drugs and viruses: mechanistic similarities and implications for AAV gene therapy . Clin. Pharmacol. Ther. 112 : 751 – 753 . 10.1002/cpt.2500 PubMedGoogle Scholar Chand , D. , Mohr , F. , McMillan , H. et al. ( 2021 ). Hepatotoxicity following administration of onasemnogene abeparvovec (AVXS-101) for the treatment of spinal muscular atrophy . J. Hepatol. 74 ( 3 ): 560 – 566 . 10.1016/j.jhep.2020.11.001 CASPubMedWeb of Science®Google Scholar Galletta , F. , Cucinotta , U. , Marseglia , L. et al. ( 2022 ). Hemophagocytic lymphohistiocytosis following gene replacement therapy in a child with type 1 spinal muscular atrophy . J. Clin. Pharm. Ther. 47 ( 9 ): 1478 – 1481 . 10.1111/jcpt.13733 CASPubMedGoogle Scholar Flanigan , K.M. , Campbell , K. , Viollet , L. et al. ( 2013 ). Anti-dystrophin T cell responses in Duchenne muscular dystrophy: prevalence and a glucocorticoid treatment effect . Hum. Gene Ther. 24 ( 9 ): 797 – 806 . 10.1089/hum.2013.092 CASPubMedGoogle Scholar Leavitt , A.D. , Konkle , B.A. , Stine , K. et al. ( 2020 ). Updated follow-up of the Alta study, a phase 1/2 study of Giroctocogene Fitelparvovec (SB-525) gene therapy in adults with severe hemophilia a . Blood 136 . 10.1182/blood-2020-137648 Web of Science®Google Scholar Ertl , H.C.J. ( 2019 ). Preclinical models to assess the immunogenicity of AAV vectors . Cell. Immunol. 342 : 103722 . 10.1016/j.cellimm.2017.11.006 PubMedGoogle Scholar Evers , M. , de Haan , M. , Valles-Sanchez , A. et al. ( 2019 ). Translating preclinical data to a human equivalent dose for AMT-130 AAV gene therapy for early manifest Huntington's disease . Mov. Disord. 34 : S7 – S8 . PubMedWeb of Science®Google Scholar Rodrigues , F.B. and Wild , E.J. ( 2020 ). Huntington's Disease clinical trials corner: April 2020 . J. Huntingtons Dis. 9 ( 2 ): 185 – 197 . 10.3233/JHD-200002 PubMedWeb of Science®Google Scholar ( 2019 ). Safety and Proof-of-Concept (POC) Study With AMT-130 in Adults With Early Manifest Huntington Disease . ClinicalTrials.gov Identifier: NCT04120493. Available from: https://clinicaltrials.gov/ct2/show/NCT04120493 . Google Scholar EMA/CHMP/571076/2022: assessment report for Upstaza, International non-proprietary name: eladocagene exuparvovec ( 2022 ). Available from: https://www.ema.europa.eu/en/documents/assessment-report/upstaza-epar-public-assessment-report_en.pdf . Google Scholar U.S. Food and Drug Administration ( 2019 ). ZOLGENSMA (onasemnogene abeparvovec-xioi): highlights of prescribing information . Available from: https://www.fda.gov/media/126109/download . Google Scholar Spronck , E.A. , Liu , Y.P. , Lubelski , J. et al. ( 2019 ). Enhanced factor IX activity following administration of AAV5-R338L "Padua" factor IX versus AAV5 WT human factor IX in NHPs . Mol. Ther. Methods Clin. Dev. 15 : 221 – 231 . 10.1016/j.omtm.2019.09.005 CASPubMedGoogle Scholar uniQure and CSL Behring Announce Primary Endpoint Achieved in HOPE-B Pivotal Trial of Etranacogene Dezaparvovec Gene Therapy in Patients with Hemophilia B . ( 2021 ). Available from: https://www.cslbehring.com/newsroom/2021/hope-b-gene-therapy-for-hemophilia-b-topline-results . Google Scholar Bunting , S. , Zhang , L. , Xie , L. et al. ( 2018 ). Gene therapy with BMN 270 results in therapeutic levels of FVIII in mice and primates and normalization of bleeding in hemophilic mice . Mol. Ther. 26 ( 2 ): 496 – 509 . 10.1016/j.ymthe.2017.12.009 CASPubMedWeb of Science®Google Scholar Long , B.R. , Sandza , K. , Holcomb , J. et al. ( 2019 ). The impact of pre-existing immunity on the non-clinical pharmacodynamics of AAV5-based gene therapy . Mol. Ther. Methods Clin. Dev. 13 : 440 – 452 . 10.1016/j.omtm.2019.03.006 CASPubMedGoogle Scholar Rangarajan , S. , Walsh , L. , Lester , W. et al. ( 2017 ). AAV5-factor VIII gene transfer in severe hemophilia A . N. Engl. J. Med. 377 ( 26 ): 2519 – 2530 . 10.1056/NEJMoa1708483 CASPubMedWeb of Science®Google Scholar Ozelo , M.C. , Mahlangu , J. , Pasi , K.J. et al. ( 2022 ). Valoctocogene Roxaparvovec gene therapy for hemophilia A . N. Engl. J. Med. 386 ( 11 ): 1013 – 1025 . 10.1056/NEJMoa2113708 CASPubMedWeb of Science®Google Scholar Meyer , K. ( 2019 ). SB-525, a novel gene therapy for treatment of hemophilia A. Presented at the NorCal SOT meeting . Available from: https://www.toxicology.org/groups/rc/NorCal/docs/NorCal-SOT-2019-Fall-Symposium-Meyer-Presentation.pdf . Google Scholar Yasuda , M. , Huston , M.W. , Pagant , S. et al. ( 2020 ). AAV2/6 gene therapy in a murine model of fabry disease results in supraphysiological enzyme activity and effective substrate reduction . Mol. Ther. Methods Clin. Dev. 18 : 607 – 619 . 10.1016/j.omtm.2020.07.002 CASPubMedGoogle Scholar Sangamo Investor Presentation ( May 2022 ). Available from: https://investor.sangamo.com/static-files/79ddb512-5379-4537-8e55-1e8903e9763d . Google Scholar U.S. Food and Drug Administration ( 2020 ). Human gene therapy for hemophilia: guidance for industry . Available from: https://www.fda.gov/media/113799/download . Google Scholar U.S. Food and Drug Administration ( 2021 ). Human gene therapy for neurodegenerative diseases: draft guidance for industry . Available from: https://www.fda.gov/media/144886/download . Google Scholar U.S. Food and Drug Administration ( 2020 ). Human gene therapy for rare diseases: guidance for industry . Available from: https://www.fda.gov/media/113807/download . Google Scholar Chen , N. , Sun , K. , Chemuturi , N.V. et al. ( 2022 ). The perspective of DMPK on recombinant adeno-associated virus-based gene therapy: past learning, current support, and future contribution . AAPS J. 24 ( 1 ): 31 . 10.1208/s12248-021-00678-7 PubMedWeb of Science®Google Scholar Von Drygalski , A. , Giermasz , A. , Castaman , G. et al. ( 2019 ). Etranacogene dezaparvovec (AMT-061 phase 2b): normal/near normal FIX activity and bleed cessation in hemophilia B . Blood Adv. 3 ( 21 ): 3241 – 3247 . 10.1182/bloodadvances.2019000811 PubMedWeb of Science®Google Scholar Fong , S. , Yates , B. , Sihn , C.R. et al. ( 2022 ). Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A . Nat. Med. 28 ( 4 ): 789 – 797 . 10.1038/s41591-022-01751-0 CASPubMedWeb of Science®Google Scholar Der , K. , Wada , R. , Huston , M. et al. ( 2022 ). ST-920 translational pharmacokinetic–pharmacodynamic (PK/PD) model from mouse to human in Fabry disease . In: Poster #066 Presented at the 18th Annual WORLDSymposium, February 2022, San Diego, CA, USA . Google Scholar Narula , J. , Luo , H.B. , Ko , G. et al. ( 2022 ). A quantitative systems pharmacology (QSP) model for design and species-translation of bio-distribution studies of AAV-based gene therapies . Mol. Ther. 30 ( 4 ): 199 – 200 . Web of Science®Google Scholar Sun , K.F. , Zhang , Z.W. , Ko , G. et al. ( 2021 ). Physiologically based pharmacokinetic modeling for the biodistribution of adeno-associated virus serotype 8 after intravenous administration in mice and non-human primates . Mol. Ther. 29 ( 4 ): 129 – 130 . Web of Science®Google Scholar Fong , S. , Handyside , B. , Sihn , C.R. et al. ( 2020 ). Induction of ER stress by an AAV5 BDD FVIII construct is dependent on the strength of the hepatic-specific promoter . Mol. Ther. Methods Clin. Dev. 18 : 620 – 630 . 10.1016/j.omtm.2020.07.005 CASPubMedGoogle Scholar Roctavian: EPAR – Product Information . ( 2022 ). Available from: https://www.ema.europa.eu/documents/product-information/roctavian-epar-product-information_en.pdf . Google Scholar Verdera , H.C. , Kuranda , K. , and Mingozzi , F. ( 2020 ). AAV vector immunogenicity in humans: a long journey to successful gene transfer . Mol. Ther. 28 ( 3 ): 723 – 746 . 10.1016/j.ymthe.2019.12.010 CASPubMedWeb of Science®Google Scholar Nathwani , A.C. , Reiss , U. , Tuddenham , E. et al. ( 2018 ). Adeno-associated mediated gene transfer for hemophilia B:8 year follow up and impact of removing "Empty Viral Particles" on safety and efficacy of gene transfer . Blood 132 ( Suppl 1 ): 491 . 10.1182/blood-2018-99-118334 Web of Science®Google Scholar Leebeek , F.W.G. , Meijer , K. , Coppens , M. et al. ( 2021 ). Amt-060 gene therapy in adults with severe or moderate-severe hemophilia B confirm stable fix expression and sustained reductions in bleeding for up to 5 years . Haemophilia 27 : 41 . PubMedWeb of Science®Google Scholar Mendell , J.R. , Al-Zaidy , S.A. , Lehman , K.J. et al. ( 2021 ). Five-year extension results of the phase 1 START trial of Onasemnogene Abeparvovec in spinal muscular atrophy . JAMA Neurol. 78 ( 7 ): 834 – 841 . 10.1001/jamaneurol.2021.1272 PubMedWeb of Science®Google Scholar Drug Development for Gene Therapy: Translational Biomarkers, Bioanalysis, and Companion Diagnostics ReferencesRelatedInformation

The anti-tumor efficacy of different FGFR2-ADC BAY 1187982 treatment schedules on MFM-223 human TNBC mouse model.

Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.

The FGFR2-ADC BAY 1187982 inhibits microtubule polymerization resulting in induction of apoptosis.

The expression of FGFR2 on tumor cell surface and half maximal inhibitory concentration of cell viability for FGFR2-ADC BAY 1187982 and control ADC.

Supplementary Materials and Methods and Supplementary Figure Legends

Immunogenicity has imposed a challenge to efficacy and safety evaluation of adeno-associated virus (AAV) vector-based gene therapies. Mild to severe adverse events observed in clinical development have been implicated with host immune responses against AAV gene therapies, resulting in comprehensive evaluation of immunogenicity during nonclinical and clinical studies mandated by health authorities. Immunogenicity of AAV gene therapies is complex due to the number of risk factors associated with product components and pre-existing immunity in human subjects. Different clinical mitigation strategies have been employed to alleviate treatment-induced or -boosted immunogenicity in order to achieve desired efficacy, reduce toxicity, or treat more patients who are seropositive to AAV vectors. In this review, the immunogenicity risk assessment, manifestation of immunogenicity and its impact in nonclinical and clinical studies, and various clinical mitigation strategies are summarized. Last, we present bioanalytical strategies, methodologies, and assay validation applied to appropriately monitor immunogenicity in AAV gene therapy-treated subjects.

In this manuscript, the European Bioanalysis Forum reports back on their discussions on practical and scientific considerations related to bioanalytical applications of quantitative polymerase chain reaction. This publication follows an earlier publication in which the European Bioanalysis Forum recommends to consider principles of context of use when defining assay acceptance criteria for method validation criteria and sample analysis.

Background Gene therapy for hemophilia A has the potential to reduce the treatment burden for patients and their care providers by eliminating the need for regular factor VIII (FVIII) prophylaxis through long-term expression of endogenous FVIII at levels sufficient to provide bleed protection. Host immunity to the capsid serotype limits patients' eligibility and may impact the balance between vector dose and clinical outcome. BAY 2599023 (AAVhu37FVIII) is the first clinical-stage adeno-associated virus (AAV) gene therapy vector based on the AAVhu37 serotype. BAY 2599023 is a non-replicating AAV vector and contains a single-stranded DNA genome encoding a B-domain-deleted FVIII, under the control of a liver-specific promoter/enhancer combination optimized for transgenic expression. The AAVhu37 capsid is a member of the hepatotropic clade E family, and was selected based on preclinical studies demonstrating efficient liver-directed FVIII gene transfer, favorable biodistribution and durable FVIII expression. However, pre-existing humoral immunity against AAV capsids may limit patient eligibility. Here, we evaluate the seroprevalence and titer levels of pre-existing neutralizing antibodies (Nabs) against AAVhu37, and additional AAV capsids, using cell-based transduction inhibition assays. We also report current, preliminary, long-term safety and FVIII activity following a single intravenous infusion of BAY 2599023, in a phase 1/2 open-label, first-in-human, dose-finding study (NCT03588299). Methods Seroprevalence and titer distribution of Nabs against AAVhu37, AAV5 and AAV8 have been assessed in serum samples derived from 100 US patients with hemophilia A (African American and Caucasian male donors, 19-61 years). For AAVhu37, the clinical trial Nab assay was utilized to determine Nab titer levels according to cellular transduction inhibition. Additionally, we developed and fully validated Nab assays for AAV5 and AAV8. The ongoing BAY 2599023 phase 1/2 dose-finding study included male patients aged ≥18 years with severe hemophilia A, each receiving a single intravenous infusion of BAY 2599023. Patients were enrolled sequentially into three dose cohorts (0.5 × 1013 GC/kg, 1.0 × 1013 GC/kg and 2.0 × 1013 GC/kg), each comprising two patients. Patients had no history of FVIII inhibitors, no detectable neutralizing immunity against the AAVhu37 capsid above a Nab titer of 1:5, and ≥150 exposure days to FVIII products. Primary endpoints were adverse events (AEs), serious AEs (SAEs) and AEs/SAEs of special interest (S/AESIs). The secondary endpoint was change in FVIII activity from baseline. Informed patient consent and ethics committee approval were obtained. Results In the seroprevalence study, the lowest pre-existing Nab prevalence was found for AAVhu37, with a low maximum observed titer of 1:26. Based on our results, 86% of patients would be eligible for AAVhu37-based treatment (Table 1). To date, patients in the first (0.5 × 1013 GC/kg) and second cohort (1.0 × 1013 GC/kg) have completed ≥52 weeks of observation; patients in cohort 3 (2.0 × 1013 GC/kg) have at least 33 weeks of observation. Regardless of the level achieved and the assay used, 5 out of 6 of patients show sustained FVIII levels (all ≥5%) over time and up to 16 months. Patients in cohorts 2 and 3 have all been off prophylaxis since ~6 weeks after gene transfer. No spontaneous bleeds were reported after achieving protective FVIII levels (>15 IU/dL) and discontinuation of prophylaxis in the third cohort. No SAEs have been reported to date. Mild-to-moderate elevation in alanine aminotransferase/aspartate aminotransferase were recorded for one patient in the second cohort and both patients in the third cohort. All were treated with corticosteroids (one resolved, two in resolution). The latest follow-up data for up to 22 months will be presented. Conclusions BAY 2599023 has a broad patient eligibility due to low seroprevalence and low titers of pre-existing Nabs against AAVhu37 compared with other AAVs. BAY 2599023 has a good safety profile, with the potential to achieve endogenous expression of FVIII at therapeutic levels over an extended period. Successful proof-of-concept has been achieved, with measurable and sustained expression of endogenous FVIII. Disclosures Pipe: Apcintex, Bayer, BioMarin, Catalyst Biosciences, CSL Behring, HEMA Biologics, Freeline, Novo Nordisk, Pfizer, F. Hoffmann-La Roche Ltd/Genentech, Inc., Sangamo Therapeutics, Sanofi, Takeda, Spark Therapeutics, uniQure: Consultancy; Siemens: Research Funding; Medical and Scientific Advisory Council to the National Hemophilia Foundation; Medical Advisory Board to World Federation of Hemophilia: Membership on an entity's Board of Directors or advisory committees. Ferrante:Bayer: Current Employment. Reis:Bayer: Current Employment. Wiegmann:Bayer: Current Employment. Lange:Bayer: Current Employment, Current equity holder in private company. Braun:Bayer: Current Employment, Current equity holder in private company. Michaels:Bayer: Current Employment.

Historically, ligand-binding assays for pharmacokinetic samples employed duplicate rather than singlet-based analysis. Herein, the Translational and absorption, distribution, metabolism and excretion (ADME) Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) presents a study aiming to determine the value of duplicate versus singlet-based testing. Based on analysis of data collected from eight organizations for 20 drug candidates representing seven molecular types and four analytical platforms, statistical comparisons of validation and in-study quality controls and study unknown samples demonstrated good agreement across duplicate sets. Simulation models were also used to assess the impact of sample duplicate characteristics on bioequivalence outcomes. Results show that testing in singlet is acceptable for assays with %CV <= 15% between duplicates. Singlet-based approach is proposed as the default for ligand-binding assays while a duplicate-based approach is needed where imprecision and/or inaccuracy impede the validation of the assay.

Abstract The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody–drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line–based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(21); 6331–9. ©2016 AACR.

Determination of concentration-time profiles in cynomolgus monkeys of a therapeutic monoclonal antibody against a soluble target revealed a substantial discrepancy between a generic anti-human IgG capture/detection and target bridging assay with the target bridging assay leading to dose- and time-dependent underquantification of drug concentrations, lack of parallelism and subsequently different pharmacokinetic parameters. In contrast, plasma levels derived from a target capture and an anti-idiotypic antibody bridging assay were in close concordance with the generic assay and demonstrated parallelism with high precision across several dilutions. The results provide a practical attempt to overcome nonparallelism by employing alternative assay formats utilizing tailored assay reagent combinations in order to obtain unbiased pharmacokinetic data.

Abstract Antibody-drug conjugates (ADC) represent a promising therapeutic approach for treatment of cancer. We have developed a novel ADC directed against fibroblast growth factor receptor 2 (FGFR2). FGFR2 is overexpressed in several cancer indications, such as gastric, breast, and ovarian cancer and thus represents a potential therapeutic target for treatment of FGFR2-positive cancer patients with ADC-based therapy. FGFR2-ADC consists of a fully human anti-FGFR2-Ab (BAY 1179470) conjugated via a stable linker to a novel auristatin cytotoxic agent licensed from Seattle Genetics. FGFR2-ADC exhibits low nM to sub-nM potency in vitro in a panel of FGFR2-positive cancer cells lines (SNU-16, MFM-223, NCI-H716) while being inactive against FGFR2-low or -negative cell lines (MDA-MB-231, HEK-293, BaF/3) and highly selective versus a control ADC. FGFR2-ADC is highly efficacious in monotherapy and results in tumor growth inhibition in the gastric cancer xenograft model SNU-16 and tumor regression in the breast cancer xenograft model MFM-223. FGFR2-ADC induces tumor stasis in the colorectal cancer xenograft model NCI-H716 and regrown tumors are sensitive to a second treatment cycle of FGFR2-ADC. FGFR2-ADC shows high efficacy in vivo in monotherapy in patient-derived (PDX) FGFR2-positive murine xenograft models, e.g. in the ovarian cancer model OV30-0511A. FGFR2-ADC is also efficacious in the gastric cancer PDX model GC10-0608 and the breast cancer model MAXF857. The toxophore metabolite of FGFR2-ADC was more than 30-fold enriched in tumors versus other organs (liver, spleen, kidneys) in NCI-H716 tumor-bearing mice. Based on the preclinical efficacy, PK and tolerability data, evaluation of FGFR2-ADC in cancer patients appears warranted. A Phase I study is planned. Citation Format: Anette Sommer, Charlotte Kopitz, Christoph Schatz, Ruprecht Zierz, Joachim Schuhmacher, Sabine Wittemer-Rump, Klaas Prins, Manuela Braun, Frank Reetz, Bertolt Kreft, Hung T. Huynh, Karl Ziegelbauer. Preclinical anti-tumor efficacy of FGFR2-ADC BAY 1187982 in patient-derived gastric, breast and ovarian cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1684. doi:10.1158/1538-7445.AM2015-1684

Abstract BAY 1187982 is an antibody drug conjugate (ADC) directed against fibroblast growth factor receptor 2 (FGFR2). FGFR2 is overexpressed in several cancer indications, such as gastric, breast, and ovarian cancer. Anti-tumor efficacy of BAY 1187982 has been demonstrated in several FGFR2-positive cancer cell line as well as patient-derived xenograft models. Toxicology findings from repeated dose preclinical safety studies in monkeys indicated effects related to the liver, kidney, heart and coagulation system. To predict the therapeutic index of BAY 1187982 in humans and to support the design of the first-in-human (FIH) study with respect to selection of dose and regimen, preclinical efficacy and toxicity findings were quantified. All available preclinical PK, TK, tumor response and toxicity data from mouse models and monkey studies were used to create a model framework to describe the PK, TK, PK/PD and TK/TD relationship as functions of BAY 1187982 dose, regimen and time. Human PK parameters based on scaling from monkey were used to predict PK profiles in humans for a range of doses and schedules. These sets of predicted exposure models were combined with the PK/PD as well as the TK/TD model to assess the expected efficacy (according to RECIST criteria) and toxicity range in humans, respectively. The dosing schedule leading to the largest therapeutic index and the dose escalation schema for the FIH study were determined. The FIH study is currently under preparation. Citation Format: Sabine Wittemer-Rump, Anette Sommer, Charlotte Kopitz, Hung Huynh, Christoph Schatz, Ruprecht Zierz, Manuela Braun, Kirstin Meyer, Dirk Laurent, Jörg Lippert, Klaas Prins. Pharmacokinetic/pharmacodynamic (PK/PD) and toxicokinetic/toxicodynamic (TK/TD) modeling of preclinical data of FGFR2-ADC (BAY 1187982) to guide dosing in phase 1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2015-1683

BACKGROUND:The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS:We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS:We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS:We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.