Using a novel cellular platform to optimize CRISPR/CAS9 technology for the gene therapy of AIDS
Despite tremendous effort devoted to the development of antiretroviral therapies to combat HIV over the past decades, AIDS remains one of the most important global infectious diseases. According to UNAIDS report on the global AIDS epidemic in 2016, the estimated number of people living with HIV rose from 7.5 million in 2010 to 36.7 million in 2015. Furthermore, drug-resistance HIV strains have recently been reported (Wensing et al., 2017). Therefore, it is important to develop new therapies to eliminate HIV in the patients. Immortalized cell lines representing the major targets of HIV in human are important for HIV research and therapeutic development. In this context, HIV mainly targets macrophage and CD4+ T lymphocytes in vivo (Iordanskiy et al., 2013). In addition, the co-receptors CCR5 and CXCR4 are required for the HIV infection of T cells (Moore et al., 1997; Zaitseva et al., 1998). Therefore, immortalized CD4+ T cell stably expressing HIV-1 co-receptor CCR5 or CXCR4 will be highly useful for HIV research. However, most of the T cell lines generally do not express adequate level of CCR5 to support the infection of CCR5 tropic HIV-1. Even if many previously established T cell lines could overcome this restriction after transduction of the expression vectors (Wu et al., 2002; Krowicka et al., 2008), the random integration in these lines leads to the unstable expression of transgene and also could affect the expression of the nearby genes (Modlich et al., 2005; Nienhuis et al., 2006).
Recently, CRISPR/CAS9 technology has become a powerful tool for efficient gene editing by inducing DNA double-strand breaks at the designated gene locus to stimulate gene mutation or homologous recombination (Hsu et al., 2014). Due to its high specificity and low off-target mutation rate (Sander and Joung, 2014; Veres et al., 2014), CRISPR/CAS9 technology is powerful for gene editing in human cells. Therefore, we employed CRISPR/CAS9 technology to develop a novel CCR5-expressing CD4+ T cell line by inducing the expression of CCR5 from its endogenous locus by inserting the CAG promoter into the promoter of the CCR5 gene via homologous recombination. The resulting cell line is permissive for HIV infection and is useful for developing gene therapy of AIDs.
The main purpose to generate Jurkat-KI-R5 cells is to create a permissive cell line for HIV infection. Therefore, we examined the susceptibility of Jurkat-KI-R5 cell to the infection of CCR5 tropic HIV. In contrast to the Jurkat cells that produce no detectable p24 antigens in the supernatant after HIV, high levels of p24 antigen were detected in the supernatant of Jurkat-KI-R5 cells after 3 days and 7 days of HIV infection (Fig. 1F). Therefore, Jurkat-KI-R5 cells represent a much-needed cellular platform to support future HIV research.
One of the key bottlenecks for HIV research is the lack of HIV permissive human cell lines. Taking advantage of the CRISPR/CAS9 gene editing technology, we generated a human T cell line that stably expresses CD4 and both co-receptors CCR5 and CXCR4. This T cell line is different from previously reported CCR5 transgenic T cell line because the CCR5 gene is expressed from its endogenous locus. The feasibility to use Jurkat-KI-R5 cells for HIV research is further supported by the findings that these cells are highly susceptible for HIV infection. Jurkat-KI-R5 cells can be useful in many aspects of HIV research and therapy development, such as HIV drug resistance, efficacy of new antiretroviral therapy, and gene therapy. In support of this notion, we used the Jurkat-KI-R5 cells to evaluate the efficiency of CCR5 disruption with CRISPR/CAS9 gene editing technology. Our data demonstrate that both CRISPR/Cas9 and CRISPR/Cas9n can efficiently ablate CCR5 with paired gRNAs in T cell lines. In this context, it remains difficult to disrupt CCR5 gene in the primary human CD4+ T cells with single gRNA (Mandal et al., 2014). As reported previously (Ran et al., 2013), paired nicking can reduce off-target activity by 50 to 1,500-fold in cell line. This could improve the safety of this technology for clinical application. The chemically synthesized and stabilized gRNA/mRNA can further increase the efficiency of CCR5 disruption. Considering that CRISPR/CAS9 gene editing technology could induce off-target genomic mutations, the Jurkat-KI-R5 cells can be further employed to improve the efficiency and safety of CRISPR/Cas9 mediated disruption of the CCR5 gene.
We thank the UCSD Translational Virology Core for HIV infection study. This work was supported by grants from Chinese Ministry of Science and Technology (2015AA020310), the National Natural Science Foundation of China (Grant Nos. 815300045 and 81373166), Shenzhen Municipal Science and Technology Innovation Council (20140405201035), and the Development and Reform Commission of Shenzhen, and a scholarship from Her Royal Highness Princess Maha Chakri Sirindhorn.
Jingjin He, Thanutra Zhang, and Xuemei Fu declare that there is no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors.
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