CRISPR based editing of SIV proviral DNA in ART treated non-human primates

Abstract

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.

Fig. 1. Construction, map and confirmation of the CRISPR-Cas9 construct targeting SIV proviral DNA in vitro.

a Schematic presentation of the full-length SIVmac239 genome (GenBank: M33262.1) with positions of targeted sites, predicted cleavage events and gRNAs sequences. b Map of AAV pX601-SaCas9-2xgRNA plasmid. c Confirmation of the expression of the plasmid SaCas9 and gRNAs. RT-PCR analysis of SaCas9 and gRNA expression in HEK293T cells transfected with the plasmid. d Gel electrophoresis analysis of PCR reaction for detection of SIV DNA after the treatment of cells with AAV9 delivering CRISPR-Cas9. Results from the PCR analysis showed a clear band of 465 bp in size in the cells that received the CRISPR-Cas9 by infection with AAV9 expressing CRISPR-Cas9. The percent excision efficiency (Efficiency (%)) in vitro shown under the PCR was calculated by quantification of the excised band (trunc.) divided by the  sum of the full-length (FL) plus the excised bands  times 100%. e Results from the PCR analysis showed a clear band of 358 bp in size in the cells that received the CRISPR-Cas9 by infection with AAV9 expressing CRISPR-Cas9. f Results from the sequencing of the 465 bp amplicon showed the breakpoint of the viral DNA, where the truncated 5′LTR is joined to the residual Gag gene after the removal of the 1014 bp DNA. g Results from the sequencing of the 358 bp amplicon showed the breakpoint of the viral DNA, where the truncated gag is joined to the residual 3′LTR after the removal of the 8447 bp DNA. Full sequencing data are available in the source file data provided with this paper. Source data are provided as a Source Data file.

Fig. 2. Excision of SIVmac239 DNA by CRISPR-Cas9 in the blood of SIV-infected rhesus macaque monkeys.

a Chinese rhesus macaques were i.v. inoculated with SIVmac239 and given a daily ART administration of tenofovir disoproxil fumarate, emtricitabine and dolutegravir, s.q. starting at 78 days post infection. Three animals (KM77, KP43 and KV88) were given 10¹³ GC/kg, i.v. and 3 weeks later underwent necropsy. One animal served as the no CRISPR control (KK09). b Plasma viral loads of the SIV-infected ART-treated rhesus macaques. The threshold of the assay was 83 copies/ml. The animals in solid black symbols and lines received AAV9/CRISPR/Cas9. The open black triangle with a dotted line (KK09) was sacrificed as no AAV9/CRISPR control. The gray symbols and lines were animals that were only used in the ex vivo screening. All viral loads were done in duplicate per time point. c AAV9-mediated delivery of CRISPR/Cas9 to ex vivo PBMCs from SIVmac239-infected animals was able to excise SIV viral DNA. Truncated 5′LTR-gag (465 bp) and gag-3′LTR (358 bp) amplicons were detected in lanes with AAV-9 CRISPR/Cas9 (+) but not in the untransformed (−). The percent excision efficiency ex vivo (Efficiency (%)) shown under the PCR was calculated by quantification of the excised band (Trunc.) divided by the  sum of the  full-length band (FL)  plus the excised bands times 100% (see Supplementary Fig. S1 for all animals). d Expression was verified by the presence of SaCas9 mRNA (547 bp), LTR and Gag gRNA scaffolds (94 bp). e Similarly, in vivo excision was confirmed in the blood of KM77 and KP43 by the PCR amplification and detection of the trunc 5′LTR to gag (465 bp) and the gag-3′LTR (358 bp) (see Supplementary Fig. S3 for KV88). The percent excision efficiency (Efficiency (%)) in vivo shown under the PCR was calculated as above. KK09 did not receive CRISPR so no efficiency was calculated. f Expression was verified by the presence of SaCas9 mRNA (547 bp), LTR and Gag gRNA scaffolds (94 bp). g Representative Sanger sequence tracings of 5′LTR-Gag (left) and Gag-3′LTR (right) CRISPR-Cas9 induced truncated SIV-specific amplicons. Target sites are highlighted in green, PAMs motifs in red, the double cleaved/end-joined site is shown as a breaking point in red. Full sequencing data are available in the source file data provided with this paper. Source data are provided as a Source Data file.

Fig. 3. Biodistribution of CRISPR-Cas9 vector in tissues.

a ddPCR analysis of Cas9 transgene DNA levels in genomic DNA extracted from various tissues of AAV9-CRISPR-SIV-treated animals (KM77, KP43 and KV88). b Detection of Cas9 RNA as a ratio to β-actin in various tissues of three animals i.v. inoculated with AAV9-CRISPR. Fifty nanograms of cDNA was analyzed in each reaction well for measuring the expression of SaCas9. β-Actin as reference gene for copy number reference reactions. Data in panels (a) and (b) are shown as mean ± SEM. Number of samples for each tissue is as follows: n = 1 for liver, tonsil and bone marrow; n = 12 for gut; n = 8 for lymph node; n = 6 for spinal cord; n = 9 for brain; n = 3 for spleen; n = 4 for heart; n = 2 for nerve, kidney, and lung. All samples were run in duplicate. c Using DNAScope technology, Cas9 DNA was detected in the mesenteric lymph nodes and spleens of SIV-infected ART-treated rhesus macaques with in vivo AAV-9-CRISPR administration (KP43, KM77 and KV88), but not in the animal without in vivo AAV-9-CRISPR (KK09). Cas9 DNA was quantified using Keyence BZ-X700 Microscope and accompanying Batch Analysis Software to determine the average area of positive signal from 30 nonoverlapping ×10 images. Data are presented as mean values of the 30 counts ±SEM. Representative images are ×10 with inserts at ×40 magnification. BDL below detection limit. d Cas9 RNA in tissues from rhesus macaques treated with AAV9-CRISPR in vivo. Using RNAScope technology, Cas9 RNA was detected in the mesenteric lymph nodes (Mes LN) and spleens of SIV-infected ART-treated rhesus macaques with in vivo AAV-9-CRISPR administration (KP43, KM77 and KV88), but not in the animal without in vivo AAV-9-CRISPR (KK09). Cas9 RNA was quantified using Keyence BZ-X700 Microscope and accompanying Batch Analysis Software to determine the average area of positive signal from 30 nonoverlapping ×10 images. Data are presented as mean values of the 30 counts ±SEM. Representative images are ×10 with inserts at ×40 magnification. BDL below detection limit. Scale bar = 200 μm Source data are provided as a Source Data file.

Fig. 4. Successful SIV viral excision in spleen, lung and lymph nodes and several areas of other tissues as indicated from in vivo AAV-9-CRISPR-treated rhesus macaques.

a ddPCR for SIV proviral DNA was performed before in vivo AAV-9-CRISPR treatment in LN biopsies and in a lymph node at necropsy. The percent decrease in SIV proviral DNA in LNs after in vivo AAV-9-CRISPR treatment was compared to SIV proviral DNA in LN biopsies prior to CRISPR. There was a greater percent decrease in KM77, KP43 and KV88 (CRISPR) vs. KK09 (control). In vivo excision was confirmed in lymph nodes of the CRISPR-treated animals by the PCR amplification and detection of the trunc 5′LTR to gag (268 bp). No excision was detected in KK09. b−f In vivo excision was confirmed in tissue (right and left lung (b), dorsal root ganglia (DRG), spinal cord and testes (c), head and body of spleen (d), brain and tonsil (e) and gut (f)) of the CRISPR-treated animals by the PCR amplification and detection of the trunc 5′LTR to gag (268 bp). No excision was detected in KK09. The percent excision efficiency in vivo (Efficiency (%)) shown under the PCR was calculated by quantification of the excised band (Trunc.) divided by the  sum of the  full-length band (FL) plus the excised  bands  times 100%. Source data are provided as a Source Data file.