Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

Abstract

Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.

Keywords: Cas9 ribonucleoprotein; HIV; gesicle.

Figure 1

Production and Characterization of Cas9 Gesicles (A) HEK293FT producer cells were transfected with the gesicle packaging mix containing CherryPicker Red, VSV-G, Cas9, and a chosen gRNA. Gesicles were released over the course of 48 hr and were collected by media filtration and ultracentrifugation. (B) Live-cell images of HEK293FT cells either untreated (−) or transfected with the gesicle packaging mix (+). The gesicle packaging mix condition showed strong CherryPicker Red fluorescence in comparison with the untreated condition. Cell lysates were prepared from HEK293FT producer cells from untreated (−) and gesicle packaging mix (+) conditions, and a western blot was run to identify protein expression of CherryPicker Red (C), VSV-G (D), and Cas9 (E). Additionally, media were taken and concentrated to obtain gesicles from untransfected (−) or gesicle packaging mix (+) cells (Conc. Media column). We observed an increased expression of CherryPicker Red (B), VSV-G (C), and Cas9 (D) in the preparation from cells transfected with the gesicle packaging mix. A parallel preparation of gesicles was prepared using the gesicle packaging mix without the presence of A/C heterodimerizer. We observed CherryPicker Red protein expression in all preparation conditions using the gesicle packaging mix (F). The absence of A/C heterodimerizer significantly decreases the expression of Cas9 as observed by western blot (G) and densitometry (H). Data are the mean + SEM of three experiments, analyzed using a one-way ANOVA with post hoc test; *p < 0.05 versus no A/C treatment group.

Figure 2

Assaying Gesicle Size and Concentration (A and B) Media from untreated HEK293FT producer cells or cells transfected with either the gesicle packaging mix or Lipofectamine were prepared in EV-depleted FBS or normal FBS and analyzed to determine size distribution and concentration of particles after ultracentrifugation and resuspension. (A) Top panels: in cells grown using EV-depleted FBS, light scatter analysis of media control from HEK293FT producer cells and producer cells transfected with the gesicle packaging mix or Lipofectamine + plasmids confirm that all conditions produce extracellular vesicles. Bottom panels: only the gesicle packaging mix and Lipofectamine conditions contain fluorescent particles. The fluorescent particles ranged between 50 and 700 nm in size. (B) Preparations of media control, gesicle packaging mix, and Lipofectamine conditions were prepared and analyzed for particle number. Transfection with the gesicle packaging mix or Lipofectamine increases the presence of extracellular vesicles in the media by 3- to 6-fold over untreated cells. Within the total population of extracellular vesicles, ∼1% are CherryPicker Red⁺ under EV-depleted conditions while normal FBS conditions exhibit only 0.38% of particles.

Figure 3

Gesicle Treatment Rapidly Delivers Protein to CHME-5 Microglia (A–G) CHME-5 microglia were treated with gesicles by centrifugation and placed on an EVOS microscope system of longitudinal imaging of live cells and CherryPicker Red fluorescence. (A) A combination of bright-field and CherryPicker Red fluorescence showed co-localization over time. Cell lysates were prepared at 1, 4, and 24 hr post-centrifugation. Cell lysates were run on a western blot and probed for CherryPicker Red (B), VSV-G (C), and Cas9 (D) in comparison with untreated cells. Densitometry was then performed for CherryPicker Red (E), VSV-G (F), and Cas9 (G). We observed a rapid yet transient delivery of Cas9 by gesicles. Data are the mean ± SEM of three experiments, analyzed using a one-way ANOVA with post hoc test; *p < 0.05 versus untreated cells.

Figure 4

Schematic of the HIV Long Terminal Repeat and Expected Products after CRISPR/Cas9 Targeting by Gesicles (A) Two gRNAs were developed to target the HIV LTR. LTR gRNA 2 targeted the NF-κB II transcription site, whereas LTR gRNA 4 targeted the TAR region. (B) The HIV provirus utilizes two LTR regions at the 5′ and 3′ ends. If CRISPR/Cas9 occurs at separate instances (denoted by the light and dark lightning bolts), mutations in these regions can occur. Black arrows denote the primer binding sites to assay mutations within the LTR regions. (C) Additionally, if a double-stranded break occurs at the same instance, there is a possibility of whole provirus excision resulting in a loss of HIV proviral copies. Black arrows denote primers used to assay the loss of integrated proviruses by ddPCR.

Figure 5

Gesicle Treatment Causes Specific Mutation Events at the HIV LTR HIV-NanoLuc CHME-5 microglia were assayed to determine mutation and a loss of proviral copy number. (A) T7E1 of the 5′ LTR amplified region showed positive products for mutation by LTR gRNA 4. (B) Droplet digital PCR was performed using a probe for the NanoLuc and the Nef regions of the provirus relative to a GGT1, a single copy gene. We observed a decrease in proviral copies per genome by LTR gRNA 4. (C) TIDE analysis was performed for LTR gRNA 4, confirming mutational events near the designated gRNA target site. The average efficiency of mutation events was 8%. (D) Two of the top off-target sites for LTR gRNA 4 were amplified and assayed for mutational events by TIDE analysis. Off-target 1 showed little to no variation from the control sample with an efficiency of 0.6%. Data are the mean ± SEM of three experiments, analyzed using a two-way ANOVA with post hoc test; *p < 0.05 versus untreated cells for NanoLuc ddPCR; ^#p < 0.05 versus untreated cells for Nef.

Figure 6

Gesicle Treatment Reduces HIV Proviral Activity (A) Cell populations treated with LTR gRNA 2 and LTR gRNA 4 were stimulated with pro-inflammatory factors, and cell lysates were taken for the luciferase assay. LTR gRNA 2 and 4 significantly decreased proviral activity after stimulation with TNF-α (50 ng/mL). Additionally, LTR gRNA 4 significantly decreased proviral activity under both basal conditions and LPS (100 ng/mL) stimulation. (C) Lysates were also run on a WES and probed for the HIV protein Nef. (B) Densitometry analysis confirms that HIV viral protein Nef is decreased using LTR gRNAs 2 and 4 after TNF-α treatment. (D) Immunohistochemistry also showed decreased immunoreactivity of Nef in cells treated with LTR gRNA 4. Data are the mean ± SEM of two stable cultures, with three experiments each, analyzed using a two-way ANOVA with post hoc test for each stimulation group (A) or one-way ANOVA with post hoc test (B). *p < 0.05 versus untreated cells: unstimulated; ^#p < 0.05 versus untreated cells: LPS stimulated; ^ˆp < 0.05 versus untreated cells: TNF-α stimulated. 2, LTR gRNA 2; 4, LTR gRNA 4; P, protamine; U, untreated.

Figure 7

Gesicle Dosage and Viability (A–D) HIV-NanoLuc CHME-5 microglia were treated with gesicles containing LTR gRNA 4 at varying concentrations (0.01–100 μg protein/mL). (A) Live-cell images 24 hr post-centrifugation indicated CherryPicker Red fluorescence increases as gesicle dose increases. (B) Viability 24 hr post-gesicle treatment was assayed using the ATP viability assay. No significant differences were detected between the different gesicle concentrations. (C) Long-term viability after cellular expansions and culture was measured by cell yield and % viability using trypan blue. No significant differences were observed between treatment conditions. (D) Cells treated with 10 and 100 μg protein/mL concentrations of LTR gRNA 4 or protamine alone. After expansion, cells were plated and stimulated with pro-inflammatory factors LPS (100 ng/mL) and TNF-α (50 ng/mL), which was repeated for 2 additional weeks. A significant decrease in proviral activity was observed using 100 μg/mL at all conditions and time points versus control samples. Cells treated with 10 μg/mL showed significant proviral inhibition after LPS and TNF-α stimulation at weeks 1 and 2, but no significant differences were observed at week 3 versus control samples. Data are the mean + SEM of three experiments (B), or three stable cultures with three experiments each (C and D), analyzed using a one-way ANOVA with post hoc test (B) or analyzed using a two-way ANOVA with post hoc test for each stimulation group (C and D); *p < 0.05 versus untreated cells: unstimulated; ^#p < 0.05 versus untreated cells: LPS stimulated; ^ˆp < 0.05 versus untreated cells: TNF-α stimulated.