Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates

Abstract

In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 10¹², 1.4 × 10¹³ and 1.4 × 10¹⁴ genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.

Fig. 1. Multiple mismatches in sites identified from genome searches using LTR or Gag guide RNAs.

All sites in the bioinformatic output from the rhesus macaque genome searches using SIV guide LTR allowing three differences and Gag allowing four differences are listed. The guide (colored) and PAM sequence (gray) are listed on top, the chromosomal sequence on the bottom with the locations and identity of mismatches shown in boxes. The two types of bulges are also displayed. Gaps in the chromosomal DNA relative the guide RNA are shown in red boxes with dashes. Additional nucleotides in the chromosomal DNA relative to the guide are shown in red triangles. The number of mismatches and bulges and match score for each chromosomal locations are listed in the right column. No loci were identified without mismatches in the proximal 8 nucleotides where differences are less tolerated, outlined in red. Moreover, none of the nominated off-target loci have a penalty match score less than 2 (right column). Bp base pair, chr chromosome, LTR long terminal repeats, MM mismatch, PAM protospacer-adjacent motif, SIV simian immunodeficiency virus.

Fig. 2. Non-human primate study design and viral loads.

Initial study design n = 10 (A), follow-up study design n = 2 (B), study chart with groups 1-4 identified (C), Individual plasma viral loads (log10 copies/mL of plasma) (D) and mean and SEM of plasma viral loads of 4 Groups (E). Black is untreated Group 1; red is Group 2, 10¹² GC/kg; light blue is Group 3, 10¹³ GC/kg; dark blue is Group 4, 10¹⁴ GC/kg. Mean and SEM are shown for the group at days post infection. ART anti-retroviral therapy, CHOP Children’s Hospital of Philadelphia, GC genome copies, IV intravenous, SIV simian immunodeficiency virus, sq subcutaneous, TCID₅₀ median tissue culture infectious dose, UNC University of North Carolina.

Fig. 3. Biodistribution of EBT-001.

EBT-001 vector DNA biodistribution as log10 copies of EBT-001 DNA per µg of monkey gDNA in major tissue reservoirs of HIV (A) and other tissues, including various brain regions (B). Symbols represent individual animals and mean and SEM are shown. gDNA, genomic DNA; SEM, standard error of the mean. Untreated 3 mos- untreated animals did not receive EBT-001 but where necropsied at the same time as the EBT-001 animals treated for 3 months. ND not determined.

Fig. 4. Proviral excision of SIV DNA in tissues and blood.

Example of 5G and G3 PCR of necropsy tissues from monkey MA285 (A). Presence of viral excision (displayed as black boxes), absence of viral excision (gray boxes), or absence of full-length and excised product (white boxes) in multiple tissues at necropsy (B). Example of 5G and G3 PCR from blood of animals BM79 and CP26 pre-EBT-001 and 4, 5, and 6 months post-EBT-001 (C). Presence of viral excision (black box) or absence of viral excision (gray boxes) from the blood of animals after EBT-001 (D). Dotted line represents the necropsy timepoint of 3 months or 6 months. No blood was obtained from the 10¹⁴ GC/kg animals at 0.5-3.0 months post-EBT-001 due to COVID shutdown. GC genome counts, mes ln mesenteric lymph node, PCR polymerase chain reaction, SIV simian immunodeficiency virus.

Fig. 5. Clinical parameters of SIV-infected ART-treated animals untreated or treated with EBT-001.

The mean (SEM) of animal’s weights are shown as percent change of pre-EBT-001 weight. Weights from EBT-001 treated (red, light blue, and dark blue lines) are significantly increased compared to untreated (black) (P = 0.029). A linear mixed-effects model was used to compare percent change in weight from pre-EBT-001 over time between treated vs untreated groups. Statistical significance was based on a 2-sided alpha level of less than 0.05 (A) The fold change in lymphocyte count from the CBC data over the average pre-EBT-001 values are shown. Each symbol is one animal and shown as the mean and SEM (B). The fold change in monocyte count from the CBC data over the average pre-EBT-001 values are shown. Each symbol is one animal and shown as the mean and SEM (C). The scatter dot plots show the mean and SEM, where each symbol represents an animal from pre-infection, pre-EBT-001 and 3 months post-EBT-001 for cholesterol (D), glucose (E), phosphorus (F), calcium (G), globin (H), total protein (I), ALP (J), ALT (K), and AST (L). Cytokines measured by MSD over time post-EBT-001 are shown for individual animals for plasma IFN-gamma (M), plasma IL-7 (N), plasma IL-15 (O). ALP alkaline phosphatase, ALT alanine transaminase, ART anti-retroviral therapy, AST aspartate transaminase, CBC complete blood count, IL interleukin, Mo month, MSD mesoscale discovery, SEM standard error of the mean, SIV simian immunodeficiency virus.