Generation of improved humanized mouse models for human infectious diseases

Abstract

The study of human-specific infectious agents has been hindered by the lack of optimal small animal models. More recently development of novel strains of immunodeficient mice has begun to provide the opportunity to utilize small animal models for the study of many human-specific infectious agents. The introduction of a targeted mutation in the IL2 receptor common gamma chain gene (IL2rg(null)) in mice already deficient in T and B cells led to a breakthrough in the ability to engraft hematopoietic stem cells, as well as functional human lymphoid cells and tissues, effectively creating human immune systems in immunodeficient mice. These humanized mice are becoming increasingly important as pre-clinical models for the study of human immunodeficiency virus-1 (HIV-1) and other human-specific infectious agents. However, there remain a number of opportunities to further improve humanized mouse models for the study of human-specific infectious agents. This is being done by the implementation of innovative technologies, which collectively will accelerate the development of new models of genetically modified mice, including; i) modifications of the host to reduce innate immunity, which impedes human cell engraftment; ii) genetic modification to provide human-specific growth factors and cytokines required for optimal human cell growth and function; iii) and new cell and tissue engraftment protocols. The development of "next generation" humanized mouse models continues to provide exciting opportunities for the establishment of robust small animal models to study the pathogenesis of human-specific infectious agents, as well as for testing the efficacy of therapeutic agents and experimental vaccines.

Keywords: Animal model; Humanized mice; Immune response; Immunodeficient mouse; Infectious agents; NSG.

Fig. 1

NSG mice provide a powerful platform for engraftment of human cells and tissues. Limitations in the development and function of certain lineages of human cells can be overcome by transgenic expression of human HLA molecules, cytokines, and other species-specific factors and by targeting mouse genes to eliminate host MHC antigens and other genes to further reduce innate immunity.

Fig. 2

NSG mice and recently developed NSG-based models can support engraftment with multiple types of human cells and tissues. These engrafted human cells support infection with many different human pathogens.

Fig. 3

Simple schematic representation of ZFN, TALEN and CRISPR modes of action. Starting with genomic DNA target sequence, nucleases are introduced into cells or oocytes. These can be, on the left ZFN or TALEN composed of a pair of proteins each designed to bind a defined 16–20 bases, each unit also carries an obligate homo or heterodimer of the FokI nuclease, which upon dimerization leads to a double stranded cut (ds-cut) in the intervening DNA spacer sequence. Or, on the right the protein Cas9 which causes a double stranded break at the target site when complexed with a signal guide RNA (sgRNA) defining a 17–20bp target sequence (plus a trinucleotide 5’NGG, protospacer adjacent motif (PAM) which is recognized by Cas9).Upon a double stranded DNA break occurring, a repair process is initiated leading to, in the absence of homologous DNA, nonhomologues end-joining (NHEJ) repair and the deletion of one, to many hundreds of bases, i.e. targeted deletion mutation; or if the presence of DNA with homology to the target cut site, homologous recombination or homologous directed repair (HDR) can occur providing precise DNA/gene sequence integration (Gaj et al., 2013; Orlando et al., 2010). Both NHEJ and HDR have been highly successful mouse in directing oocytes of multiple backgrounds, although at the time of writing the frequency of homologous recombination is general less than of NHEJ (Gaj et al., 2013; Low et al., 2014; Yang et al., 2013).

Fig. 4

Sequence of events for gene modification using targeted nucleases. A) ZFN and TALEN are introduced into fertilized oocytes generally as mRNAs encoding the binding site and nucleases (ZFN and TALEN) or for CRISPR, the Cas9 nuclease and its signal-guide RNA (sgRNA). B) Microinjected oocytes are introduced into pseudopregnant host females and carried to term. C) The resulting offspring is screened for the desired genetic modification event, generally by PCR and sequencing. D) Mice carrying desired modification events are bred to ensure germline transmission and eliminate any possible mosaicism.