Background
Rodents cannot be infected with HIV due to multiple obstacles. First, HIV requires human CD4/CCR5/CXCR4 on human immunocytes, each are structured differently in mice. Second, post-transcriptional factors absent in rodent cells limit viral RNA export, translation, stability, viral assembly, and release. Third, rodent CD8+ T-cell viral control is active in rodents with active killing of virus-infected cells with antiviral cytokine release. Attempts by others to use transgenic mouse models have all failed due to random transgene insertion and expression, and disruption of endogenous genes. These led to off-target toxicities and blocks in viral assembly. Herein, a knock-in (KI) of huCD4, huCCR5, and huC1qbp and CD8+ T-cell control resulted in stable physiologically HIV expression.
Methods
CRISPR-Cas9 homologous recombination inserted human CD4, CCR5, and C1qbp[D106G] into defined loci in the mouse genome generating physiologically relevant human expression patterns. Mouse C1qbp (p32) facilitated viral growth after KI by human specific amino acids. All site-specific CRISPR KIs were confirmed using RT-qPCR and Sanger Sequencing. CD4+ T-cells isolated from peripheral blood lymphocytes, splenocytes, thymocytes, and lymph nodes expressed human CD4 and CCR5 cell receptors in the correct orientation.
Results
Genotypic and phenotypic tests confirmed HIV receptor/co-receptor expression. FACS staining confirmed abundant huCD4⁺CCR5⁺ T-cells in the blood and peripheral lymphoid organs. HIV-1ADA (MOI = 0.1) challenge in rodent PBL led to productive viral replication. Infectivity was affirmed in vivo after intraperitoneal HIV-1ADA (105 TCID50) challenge assayed by plasma viral load (qPCR and ddPCR) and tissue viral DNA. Extracted PBLs and splenocytes from infected animals and formalin-fixed spleen affirmed HIV-1p24 antigen expression (Figure). CD8+ T-cell depletion accelerated viral infection.
Conclusions
Several viral replication blocks have been reported in mice. These include barriers to viral entry, transcription, and assembly. The CRISPR KI mouse model offers physiologically regulated expression of HIV entry receptors and represents a notable advancement over any previous transgenic system. The mouse provides a new platform for studying HIV entry, early replication, and virion production to study human therapeutic and vaccine strategies. The next-generation MHC class II CD4/CCR5 and C1qbp mice that are known to be essential for the maturation and activation of CD4+ T-cells are being bred.
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