Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study

Abstract

A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5'-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.

Figure 1. Excision of HIV-1 DNA by rAAV₉:saCas9/gRNA Tg26 MEF

A. Schematic illustration of HIV-1 highlighting the position between the Gag and Pol genes (3100 bp) that was removed to create a transgene for developing Tg26 animals. The positions of gRNAs LTR 1 and Gag D, and their nucleode compositions are shown. Red letters indicate the PAM sequence. The top left of the diagram shows the positions of the primers used for PCR (P1 and P2) and nested PCR (P1′ and P2′) with the expected amplicons before and after excision. B. Depiction of MEFs derived from Tg26 and results from gel analysis of PCR amplification of HIV-1 DNA after treatment with increasing amounts of rAAV:Cas9/gRNAs. The position of full-length (1323 bp) and the truncated (368 bp) PCR products are shown.

Figure 2. In vivo excision of HIV-1 DNA by rAAV₉:saCas9/gRNA in various tissues of Tg26 mice

A. Diagram of the procedure used for the inoculation of rAAV₉:Cas9/gRNAs and the organs used for the analysis. B. Results from PCR of DNA obtained from lines of Tg26 mice with and without rAAV₉ show the positions of 1323 bp and the truncated 368 bp in the rAAV₉ treated samples (left). Gel analysis of PCR products using two pairs of primers; P1/P2 for detection of 368 bp fragment and the nested primers P1′/P2′ for detection of the 183 bp fragment. The full-length PCR product is shown by an arrowhead(middle). Results from double primer PCR amplification of the MEF DNA again showing the expected two truncated HIV-1 DNA fragments. The position of the full-length amplicon (arrowhead) and a non-specific band (asterisk) are shown. Control (Cont.) illustrates PCR reaction in the absence of primers (right). C. Results from sequencing of the 183 bp DNA fragment shown in Panel B (middle and right panels). The positions of the primers as well as gRNAs LTR 1 and Gag D are shown. D. Results from PCR amplification of DNA from various tissues (lanes 1 and 2) and MEFs (lane 3) demonstrating the absence of the 183 bp truncated DNA fragment in animals with no inoculation of rAAV₉:saCas9/gRNAs and the detection of this band in the animals and cells that received rAAV₉:saCas9/gRNAs.

Figure 3. Elimination of segments of the integrated HIV-1 DNA from rat blood cells after inoculation with rAAV₉:saCas9/gRNA and expression of viral RNAs

A. Total DNA from circulating lymphocytes of the control (untreated) and experimental (treated with rAAV₉:saCas9/gRNA) rats was prepared and used for PCR amplification using a set of primers derived from the 5′-LTR and Gag gene (shown in Materials and Methods). After gel electrophoresis, a short DNA fragment of approximately 221 bp was detected in the treated, but not the control samples, which were then purified and cloned in a TA vector. Several clones were selected for DNA sequencing. Four representative DNA sequences (C1–C4) obtained from each animal were aligned to the reference LTR-Gag region of the HIV-1 pNL_4–1 sequence. The positions and nucletide composition of LTR 1 and Gag D target sequences are highlighted in green, PAM in red and LTR specific primers using PCR are highlighted in blue. B. Total RNA prepared from circulating lymphocytes and lymph nodes of transgenic rats, control (untreated) and treated with rAAV₉:saCas9/gRNA for 10 days were prepared and used for quantatitive RT-PCR for detection of Gag RNA and Env RNA. Expression of β-actin in each assay was determined and the values were used as reference for quantification of viral RNA expression.