Recent clinical trials using patient's own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined... Show moreRecent clinical trials using patient's own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott-Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles). Show less
Wijk, R.C. van; Krekels, E.H.J.; Kantae, V.; Ordas, A.; Kreling, T.; Harms, A.C.; ... ; Graaf, P.H. van der 2019
Zebrafish larvae are increasingly used for pharmacological research, but internal drug exposure is often not measured. Understanding pharmacokinetics is necessary for reliable translation of... Show moreZebrafish larvae are increasingly used for pharmacological research, but internal drug exposure is often not measured. Understanding pharmacokinetics is necessary for reliable translation of pharmacological results to higher vertebrates, including humans. Quantification of drug clearance and distribution requires measurements of blood concentrations. Additionally, measuring drug metabolites is of importance to understand clearance in this model organism mechanistically. We therefore mechanistically study and quantify pharmacokinetics in zebrafish larvae, and compare this to higher vertebrates, using paracetamol (acetaminophen) as paradigm compound. A method was developed to sample blood from zebrafish larvae at five days post fertilization. Blood concentrations of paracetamol and its major metabolites, paracetamol-glucuronide and paracetamol-sulphate, were measured. Blood concentration data were combined with measured amounts in larval homogenates and excreted amounts and simultaneously analysed through non-linear mixed effects modelling, quantifying absolute clearance and distribution volume. Blood sampling from zebrafish larvae was most successful from the posterior cardinal vein with median volume (interquartile range) of 1.12 (0.676-1.66) nL per blood sample. Samples were pooled (n=15-35) to reach measurable levels. Paracetamol blood concentrations at steady state were only 10% of the external paracetamol concentration. Paracetamol-sulphate was the major metabolite and its formation was quantified using a time-dependent metabolic formation rate. Absolute clearance and distribution volume correlated well to reported values in higher vertebrates, including humans. Based on blood concentrations and advanced data analysis, the mechanistic and quantitative understanding of paracetamol pharmacokinetics in zebrafish larvae has been established. This will improve the translational value of this vertebrate model organism in drug discovery and development. Show less
Zebrafish larvae (Danio rerio) are increasingly used to translate findings regarding drug efficacy and safety from in vitro-based assays to vertebrate species, including humans. However, the... Show moreZebrafish larvae (Danio rerio) are increasingly used to translate findings regarding drug efficacy and safety from in vitro-based assays to vertebrate species, including humans. However, the limited understanding of drug exposure in this species hampers its implementation in translational research. Using paracetamol as a paradigm compound, we present a novel method to characterize pharmacokinetic processes in zebrafish larvae, by combining sensitive bioanalytical methods and nonlinear mixed effects modeling. The developed method allowed quantification of paracetamol and its two major metabolites, paracetamol-sulfate and paracetamol-glucuronide in pooled samples of five lysed zebrafish larvae of 3 days post-fertilization. Paracetamol drug uptake was quantified to be 0.289 pmole/min and paracetamol clearance was quantified to be 1.7% of the total value of the larvae. With an average volume determined to be 0.290 μL, this yields an absolute clearance of 2.96 × 10(7) L/h, which scales reasonably well with clearance rates in higher vertebrates. The developed methodology will improve the success rate of drug screens in zebrafish larvae and the translation potential of findings, by allowing the establishment of accurate exposure profiles and thereby also the establishment of concentration-effect relationships. Show less
Spaink, H.; Racz, P.; Ordas, A.; Veneman, W.; Vijver, M.; Wildwater, M.; ... ; Hankemeier, T. 2015
Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap... Show moreZebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds. Show less
