Genome editing strategies: potential tools for eradicating HIV-1/AIDS

Abstract

Current therapy for controlling human immunodeficiency virus (HIV-1) infection and preventing acquired immunodeficiency syndrome (AIDS) progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells, which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or "sterile" cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS.

Figure 1. Nuclease-induced double strand breaks (DSBs) initiate the cellular homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways exploited by the ZFN, TALEN, and CRISPR genome editing technologies

Yellow squares indicate target genome. PAM, protospacer adjacent motif. Indel, insertion or deletion mutants.

Figure 2. Precise cleavage of the third nucleotide from protospacer adjacent motif (PAM) by RNA-guided Cas9 dual nucleases induces double strand breaks in the target DNA

The guide RNA (gRNA) is a chimeric stem-loop structure consisting of CRISPR RNA (crRNA) and trans-activating crRNA (tracRNA) with a GAAA tetraloop.

Figure 3. Eradication of the entire HIV-1 genome spanning between the 5′- and 3′-LTRs by Cas9/LTR-A/B gRNAs in the human U1 monocytic cell line

The integration site of the HIV-1 genome within chromosome 2 of the U1 cell line is shown. Cells were transfected with plasmids encoding Cas9 and gRNAs targeting two sequences within the 5′ and 3′ HIV-1 LTRs, called LTR-A and LTR-B. Sanger sequencing of a 1.1 kb fragment from long-range PCR using a primer pair (T492/T493) targeting the flanking sequences of the HIV-1 integration site (467 bp) validated the elimination of the entire HIV-1 genome (9,709 bp). NS, non-specific band. Similar results were seen for the second copy of the HIV-1 genome integrated into chromosome X of the U1 cell line. (See Hu et al., 2014 for a detailed description of the LTR-A and -B sequences and the results for chromosome X).

Figure 4. Stable expression of Cas9 plus LTR-A/B efficiently eradicates the HIV-1 genome in latently infected CHME5 microglial cells

Cells were transfected with plasmids expressing Cas9 and LTR-A, -B, or -A/B. After a 2-week selection with puromycin (1 g/ml), subclones were cultured by limiting dilution and single cell clones were treated with trichostatin A (TSA, 250 nM) for 2 d to induce viral reactivation before EGFP flow cytometry. A dramatic reduction in TSA-induced reactivation of latent pNL4-3- Gag-d2EGFP reporter virus was detected in most of the subclones with single or duplex LTR-gRNAs as compared with the empty pX260 control vector. (See Hu et al., 2014 for further details.)