Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.


RO
Supplemental Figure 1: Reduction of ganglionic latent HSV loads after meganuclease therapy delivered using various AAV serotypes does not depend on the route of administration.a. Experimental timeline of ocular infection and meganuclease therapy.b.HSV loads in SCGs and TGs from infected control and infected mice treated with m5 delivered by retro-orbital (RO) or whisker pad (WP) injections of 10 12 vg of various AAV.Percent decrease of HSV loads in treated mice compared to control mice and significant statistical analysis (Ordinary one-way Anova, multiple comparisons with *: p < 0.05; **: p < 0.01, ****: p < 0.0001).Exact p values are provided in the Source Data File.c, AAV loads in SCGs and TGs from infected control (CTRL, circles) and infected mice treated with m5 delivered using AAV serotype 7 (squares), 9 (upward triangles), Dj (downward triangles), Dj/8 (diamonds) administered by either RO or WP injections (see Fig. 1a).d, Percent mutation quantified by T7 assay in latent HSV genomes present in SCG and TG collected from infected control (CTRL, circles) and infected mice treated with m5 delivered using AAV serotype 7 (squares), 9 (upward triangles), Dj (downward triangles), Dj/8 (diamonds) administered by either RO or WP injections (see Fig. 1a).e-g, Same data as above in panels a-d presented per route of administration of the AAV delivery vectors.Source data are provided as a Source Data file.b-e, HSV titers in eye swabs collected daily for 3 days after the 1st (day 32 p.i. in a and day 0 in b-e), 2nd (day 39 p.i. in a and day 7 on graph b-e) or 3rd (day 46 p.i. in a and day 14 in b-e) IP injection of either vehicle (black arrows) or JQ1 (red arrows, 50 mg/kg) b, mice (n = 12) received 3 sequential IP injections of vehicle, c, mice (n = 12) received 1 IP injection of JQ1 followed by 2 sequential IP injections of vehicle, d, mice (n = 12) received 2 sequential IP injections of JQ1 followed by 1 IP injection of vehicle, and e, mice received 3 sequential IP injections of JQ1.f.Experimental timeline of ocular infection and sequential HSV reactivation with JQ1 injections.g-h, HSV viral loads in SCG (g) and TG (h) collected from mice after either 3 sequential injections of vehicle (0, n = 12), 1 JQ1 injection followed by 2 sequential injections of vehicle (1, n = 12), 2 sequential JQ1 injections followed by 1 injection of vehicle (2, n = 12), 3 sequential JQ1 injections (3, n = 12) or 7 sequential JQ1 injections (7, n = 4).ICF per square pixels

Supplemental
ICF per square pixels Supplemental figure 7. Histopathology of TG from dual meganuclease-treated mice.
Images of H&E stained trigeminal ganglia sections from either latently infected control mice not reactivated (CTRL no JQ1 (slide 10 in Supplemental  Supplemental Table 1.Virus titers in eye swabs collected after mock and JQ1 reactivations.a Virus titers are expressed in copies/ml.b Mice were subjected to 3 weekly reactivation: mock reactivation (vehicle) for Group 1, JQ1 reactivation (50 mg/kg) for Group 4 or combinations of mock (vehicle) and JQ1 (50 mg/kg) reactivations for groups 2 and 3. c At each time point, daily swabs were collected from day 0 to day 3 after each reactivation, from the left eye (L, blue) and right eye (R, orange) and analyzed separately.
Supplemental Table 3. Summary of Histologic Findings in trigeminal ganglia.
Each graph shows individual and mean values with standard deviation.Source data are provided as a Source Data file.Supplemental Figure 5 Supplemental figure 5: Histopathology of liver from dual meganuclease-treated mice.H&E staining of liver section from naive mouse (a,10x and b, 40x), HSV-infected mouse administered 3x10 12 vg AAV (c, 10x and d, 40x) and mouse administered 3x10 12 vg AAV only (e, 10x and f, 60x).Black arrows indicate hepatocellular karyomegaly, anisocytosis and anisokaryosis, red arrows indicate hepatocellular necrosis, green arrows indicate periportal or parenchymal mixed infiltrates, yellow arrow indicates pigmented Kupffer cells, blue arrow indicates suspected biliary cholestasis.Supplemental figure 6: Inflammatory cell foci in liver of meganuclease-treated mice.a, ICF in liver sections from either HSV infected control mice (control, black circles, n = 11), or treated with dual meganuclease therapy at a dose of 0.6x10 12 vg AAV (yellow circles, n= 12), 1.2x10 12 vg AAV (red circles, n = 12) and 1.8x10 12 vg AAV (purple circles n =12) from experiment described in Figure 5a-l.Statistical analysis using ordinary one-way Anova with multiple comparisons test, ns: not significant; ****: p < 0.0001.Exact p values are provided in the Source Data File.b, ICF in liver sections from either HSV infected control mice unreactivated (control no JQ1, black circles, n = 12), control mice reactivated with JQ1 (control 2x JQ1, black squares, n =12), HSV infected mice treated with dual meganuclease therapy unreactivated (AAV/MN no JQ1, open circles n =12), or reactivated with JQ1 (AAV/MN 2x JQ1, open squares n = 12) from experiment described in Figure 5m-u.Statistical analysis using unpaired one-tailed t test.ns: not significant; **: p < 0.01; ***: p < 0.001.Exact p values are provided in the Source Data File.Source data are provided as a Source Data file.
shedding after a double dose of JQ1 in 67% (6 /9) of reactivated mice.a, Latently infected mice were administered 2 IP injections of JQ1 (50 mg/kg) separated by 12h, n = 9.HSV titers in eye swabs collected from day 0 to 3 post-JQ1 are plotted for each mouse.Source data are provided as a Source Data file.quantification of AAV viral loads.a-cl, AAV loads in SCGs (a), TGs (b) and livers (c) from HSV-1 infected control (CTRL, black circles) and HSV-1 infected mice treated with AAV9-CBh-m4 (orange circles), Dj/8 (red circles), AAV9-E/CamKII-m4 (orange circles), or AAV9-E/hSyn-m4 (yellow) administered by either RO in the experiment presented in Figure7a-f.Source data are provided as a Source Data file. of m4 expression in TG from treated mice.a-b, Western blot detection of m4 expression (anti-HA) in TG collected from uninfected mice at 1 (mice ID# 1-3 and 12-14) , 4 (mice ID# 4-6 and 15-17), and 8 (mice ID# 7-10, 11, and 18-21) weeks after RO administration of 1x10 12 vg either AAV9-E/CamKII-m4 (a), or AAV9-CBh-m4 (b).d, Immune cell foci (ICF) in liver sections and e-f, AAV loads in liver (e), and TG (f) from uninfected mice treated with either AAV9-CBh-m4 black circles), or AAV9-E/CamKII-m4 (yellow circles) collected at 1, 4, and 8 weeks post AAV administration.$ Mouse ID#11 did not receive any AAV.*Sample from mice 16 and 18 are from week 4 and 8, respectively.The protein molecular weight markers were run on the same gel, but the data was acquired as a separate colorimetric data image from the chemiluminescent data image for the anti-HA (a) or anti-b-actin signals (b).Source data are provided as a Source Data file. of pscAAV plasmids.Plasmids used for the production of scAAV delivery vectors.ITR: inverted terminal repeat, mutated ITR: ITR with deletion of the D region, HA: HA-tag, nls: nuclear localization signal.

Group 1 b : vehicle -vehicle -vehicle
Number of TG per slide b Each slide had 3 sections from one TG c AAV dose administered was 1.8 x10 12 vg/mouse d Mean score = Sum of scores from TG in the group/number of TG in the group e Mean severity = Sum of scores from TG in the group/number of TG with a score > 0 in the group a