Abstract
The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3+ and CD4+ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients.
Highlights
Recombination activating gene 1 (RAG1), as well as RAG2, is required for the activation of immunoglobulin (Ig) and T cell receptor (TCR) genes by catalyzing the combinatorial joining of variable (V), diversity (D), and joining (J) gene segments that encode the antigen-recognition sequences of the Ig genes in B cells and TCR genes in the T cells, respectively [1]
A frameshift indel would introduce premature early stop codons interrupting the expression of the full-length RAG1 protein, modeling the genetic defects that fully abolish the function of RAG1 as observed in more extreme forms of severe combined immunodeficiency (SCID); it is possible that a frameshift indel in the non-core domain may still allow the alternative usage of other Mets (ATG) downstream of the indels as translation start codons to produce
SCID with a wide spectrum of disease manifestations. Both of the RAG1 and RAG2 proteins have functional domain structures that can be divided into core and non-core domains, with the core domains being the minimal region required for catalyzing V(D)J recombination and the non-core domains exerting a variety of regulatory functions, including nuclear import, interaction with other cellular proteins, and protein turnover [19]
Summary
Recombination activating gene 1 (RAG1), as well as RAG2, is required for the activation of immunoglobulin (Ig) and T cell receptor (TCR) genes by catalyzing the combinatorial joining of variable (V), diversity (D), and joining (J) gene segments that encode the antigen-recognition sequences of the Ig genes in B cells and TCR genes in the T cells, respectively [1]. Mutations in the RAG1 or RAG2 genes may lead to a failure of V(D)J recombination of Ig and TCR genes and are the prominent causes for severe combined immunodeficiency (SCID), such as Omenn syndrome, a form of primary immunodeficiency characterized by abnormal development of functional. The adaptive immune system in SCID patients is defective both in antibody response by B cells and a lack of functional T cells. These patients are highly prone to severe bacterial, viral, or fungal infections early in life and may fail to thrive. Animal models in which the RAG1 or RAG2 gene is genetically inactivated have contributed greatly to the understanding of SCID caused by the loss of function mutations in the RAG genes. We challenged the RAG1-86nt hamsters with HAdV-C6 via intranasal infection and demonstrated that these hamsters can still mount anti-adenovirus humoral immunity, though with slower kinetics in clearing the infectious viruses
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