Choosing the right mouse model: comparison of humanized NSG and NBSGW mice for in vivo HSC gene therapy.

ABSTRACT: In vivo hematopoietic stem cell (HSC) gene therapy is an emerging and promising area of focus in the gene therapy field. Humanized mouse models are frequently used to evaluate novel HSC gene therapy approaches. Here, we comprehensively evaluated 2 mouse strains, NSG and NBSGW. We studied human HSC engraftment in the bone marrow (BM), mobilization of BM-engrafted HSCs into circulation, in vivo transduction using vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vectors (VSV-G LVs), and the expression levels of surface receptors needed for transduction of viral vectors. Our findings reveal that the NBSGW strain exhibits superior engraftment of human long-term HSCs compared with the NSG strain. However, neither model resulted in a significant increase in circulating human HSCs after mobilization. We show that time after humanization as well as human chimerism levels and platelet counts in the peripheral blood can be used as surrogates for human HSC engraftment in the BM. Furthermore, we observed low expression of the low-density lipoprotein receptor, a requirement for VSV-G LV transduction, in the human HSCs present in the murine BM. Our comprehensive characterization of humanized mouse models highlights the necessity of proper validation of the model and methods to study in vivo HSC gene therapy strategies.

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NPs), which could also be used for in vivo administration, for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34+ cells. NP-mediated ex vivo delivery showed no toxicity, and the efficiency was directly correlated with the charge of the NPs. In a side-by-side comparison with electroporation, NP-mediated gene editing allowed for a 3-fold reduction in the amount of reagents, with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.