Development of an In Vitro Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release

Lengler, Johannes; Coulibaly, Sogue; Gruber, Bernhard; Ilk, Reinhard; Mayrhofer, Josef; Scheiflinger, Friedrich; Hoellriegl, Werner; Falkner, Falko G.; Rottensteiner, Hanspeter

doi:10.1016/j.omtm.2020.03.013

Cited by 14 publications

(7 citation statements)

References 29 publications

(44 reference statements)

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“…Indeed, several methods can be used to evaluate FIX activity in vitro, but the sensitivity of certain assays is very low and affected by numerous parameters such as cell density, incubation time, and culture medium. [ 32 ] This difficult evaluation of clotting activity has recently been reported by others who showed broad variations in the measurement of FIX activity levels, depending on the assay used. [ 33 ] However, in the case of severe HB, just a minor improvement in FIX levels or repopulation efficiency should be sufficient to achieve therapeutic effectiveness.…”

Section: Discussionmentioning

confidence: 98%

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

et al. 2021

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Background and Aims: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids.Approach and Results: Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and

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Section: Discussionmentioning

confidence: 98%

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

et al. 2021

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“…In HA, an approximate 1.6-fold higher FVIII:C OSA FVIII:C than CSA has been reported. 114 115 116 117 Curiously, this is the reverse of the pattern observed in some BDD-rFVIII molecules. 17 118 Rosen et al proposed that the higher OSA compared with CSA in AAV5-FVIII-SQ molecules is caused by accelerated early FXa and thrombin generation, and shorter clotting times generate higher reported FVIII activity levels.…”

Section: Fviii and Fix Measurements: Gene Therapymentioning

confidence: 81%

Factor VIII and Factor IX Activity Measurements for Hemophilia Diagnosis and Related Treatments

Bowyer

Gosselin

2022

Semin Thromb Hemost

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Accurate measurement of clotting factors VIII (FVIII) or IX (FIX) is vital for comprehensive diagnosis and management of patients with hemophilia A or B. The one-stage activated partial thromboplastin time (aPTT)-based clotting assay is the most commonly used method worldwide for testing FVIII or FIX activities. Alternatively, FVIII and FIX chromogenic substrate assays, which assess the activation of factor X, are available in some specialized laboratories. The choice of reagent or methodology can strongly influence the resulting activity. Variation between one-stage FVIII or FIX activities has been reported in the measurement of some standard and extended half-life factor replacement therapies and gene therapy for hemophilia B using different aPTT reagents. Discrepancy between one-stage and chromogenic reagents has been demonstrated in some patients with mild hemophilia A or B, the measurement of some standard and extended half-life factor replacement therapies, and the transgene expression of hemophilia A and B patients who have received gene therapy. Finally, the measurement of bispecific antibody therapy in patients with hemophilia A has highlighted differences between chromogenic assays. It is imperative that hemostasis laboratories evaluate how suitable their routine assays are for the accurate measurement of the various hemophilia treatment therapies.

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“…For the determination of in vitro biopotency, HepG2 cells were infected in duplicates with AAV8 vector carrying FIX transgene as described previously [ 42 ]. Subsequently, the chromogenic activity of FIX in the supernatant was measured using the Rox Factor IX kit (Rossix AB, Moelndal, Sweden).…”

Section: Methodsmentioning

confidence: 99%

Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM

et al. 2022

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In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method.

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Development of an In Vitro Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release

Cited by 14 publications

References 29 publications

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

Factor VIII and Factor IX Activity Measurements for Hemophilia Diagnosis and Related Treatments

Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM

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