RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Fig. 1. Transgene induction from shRNA-regulated self-silencing AAV vector using REVERSIR. a Diagram illustrating AAV transgene self-silencing using intronically-encoded shRNA and induction of expression with exogenously-administered REVERSIR. b Viral genome schematics of AAV8 GLuc reporter vector with intronic TTR shRNA and complementary (TTR shRNA/TTR-ts) or scrambled 3’ UTR target site (TTR shRNA/NT-ts) c HepG2 cells were transfected with AAV plasmids and media was collected at each timepoint. Media was fully exchanged at every collection, with each line corresponding to accumulated GLuc in supernatant from a single well since prior timepoint (n = 6 wells per condition; two-way repeated measures ANOVA with Sidak’s correction). d Recovery of reporter expression with REVERSIR in HepG2 cells. AAV constructs in (b) were co-transfected with indicated concentrations of 22-mer TTR REVERSIR or NT REVERSIR, along with a Luc2 internal control plasmid. 48 h post-transfection, GLuc and Luc2 intensities were assayed in cell supernatant and lysate, respectively. GLuc/Luc2 ratios were computed and plotted relative to TTR shRNA/NT-ts condition set to 100%. Data analyzed by one way ANOVA with Sidak’s…

Fig. 2. REVERSIR-mediated regulation of an erythropoietin transgene. a Vector genome schematics for AAV encoding mouse EPO (mEPO) transgene under control of TTR shRNA with intact (self-silencing; TTR shRNA/TTR-ts) or scrambled (non-self-silencing; TTR shRNA/NT-ts) binding site in 3’ UTR. b Mice were transduced with 2 × 1011 GC of AAV8 vectors shown in (a) for two weeks and then subcutaneously injected with REVERSIR. Serum was collected for quantification of mEPO levels by ELISA at indicated days prior to and following REVERSIR administration. c Serum EPO concentrations in mice that received 0.1 mg/kg 9-mer TTR or NT REVERSIR, compared to vehicle control on D0 (n = 4 mice for vehicle groups and n = 3 mice for REVERSIR groups). Data were analyzed by two-way repeated measures ANOVA with Tukey’s post hoc test (***p < 0.001 relative to TTR shRNA/TTR-ts + NT REVERSIR control). d EPO concentrations following treatment with increasing doses of TTR REVERSIR 2 (0.01, 0.03, 0.1, or 0.3 mg/kg). Experiments in panels (c) and (d) were conducted in parallel and compared to the same vehicle conditions. An additional control was included in (d) in which mice transduced with the non-self-silencing vector…

Fig. 3. In vivo regulation of an AAV-expressed reporter transgene by exogenous delivery of siRNA and REVERSIR. a Diagram depicting dual agent approach involving suppression of AAV transgene expression with siRNA and subsequent upregulation of protein expression by abrogation of siRNA activity with REVERSIR. b Mice were injected with 2 × 1011 GC of AAV encoding bicistronic expression of a GLuc reporter with a TTR siRNA binding site in the 3’ UTR. Mice were subcutaneously injected with vehicle or TTR siRNA at 9 mg/kg (D0). This was followed on D14 with a single molar equivalent dose of full-length 22-mer (3 mg/kg) or 9-mer TTR REVERSIR (1.6 mg/kg) and compared to NT REVERSIR control. c Serum GLuc levels normalized to pre-dose (D0) for each animal (n = 5 for PBS; n = 4 for vehicle and 22-mer TTR REVERSIR groups; n = 3 for 22-mer NT REVERSIR and both 9-mer REVERSIR groups). Data were analyzed by two-way repeated measures ANOVA followed by Tukey’s post hoc test. d Gluc transcript levels in liver tissue at D42 normalized to Gapdh control. e Serum GLuc levels at D21 in AAV-transduced mice that received TTR siRNA (9 mg/kg; D0), followed by increasing doses of 9-mer TTR REVERSIR (D14) or NT…

Fig. 4. In vitro characterization of on- and off-target activity of transgene regulator siRNA sequences. a On-target silencing efficacy (solid blue line) of three lead transgene regulator siRNA (TR-siRNA) sequences as assayed by co-transfection of serially titrated doses of siRNA with dual luciferase sensors containing a perfectly matched binding site in the 3’ UTR of Renilla luciferase (RLuc). Seed-mediated off-target repression was similarly assessed by dose-response activity of siRNA in the presence of luciferase reporters bearing either 1 (green dashed line) or 4 tandem (gray dashed line) seed-matched target sites. RLuc/FLuc ratios were normalized to the mock-transfected control (no siRNA) condition set at 100% and plotted as mean±s.e.m. Data were analyzed using ordinary one-way ANOVA with Bonferroni’s test for multiple comparisons (*p < 0.05 **p < 0.01 ***p < 0.001). b MA plots depicting differential gene expression analysis of RNA-seq data obtained from transfection of TR-siRNAs and a Tmprss6-targeting siRNA control in Hep3B cells (top; 10 nM dose harvested at 24 h) and primary mouse hepatocytes (bottom; 50 nM dose harvested at 48 h) (n = 4 biological replicates per condition).…