A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo

Abstract

Widespread use of gene therapy technologies is limited in part by the lack of small genetic switches with wide dynamic ranges that control transgene expression without the requirement of additional protein components^1-5. In this study, we engineered a class of type III hammerhead ribozymes to develop RNA switches that are highly efficient at cis-cleaving mammalian mRNAs and showed that they can be tightly regulated by a steric-blocking antisense oligonucleotide. Our variant ribozymes enabled in vivo regulation of adeno-associated virus (AAV)-delivered transgenes, allowing dose-dependent and up to 223-fold regulation of protein expression over at least 43 weeks. To test the potential of these reversible on-switches in gene therapy for anemia of chronic kidney disease⁶, we demonstrated regulated expression of physiological levels of erythropoietin with a well-tolerated dose of the inducer oligonucleotide. These small, modular and efficient RNA switches may improve the safety and efficacy of gene therapies and broaden their use.

Figure 1.. Development of a class of highly efficient hammerhead ribozymes (HHR).

(a) A diagram representing HHR-mediated inactivation of gene expression. (b) A panel of engineered HHR variants were tested in a reporter assay in which an expression vector encoding Gaussia luciferase (Gluc) and Cypridina luciferase (Cluc), each driven by an independent promoter, was used. A catalytically active ribozyme variant was placed to the 3′ UTR of the Gluc gene, and compared with a catalytically inactive form of the same ribozyme. Reporter plasmids were transfected into 293T cells, and the functional ribozyme’s activity was calculated as fold inhibition in the Gluc expression relative to the inactive ribozyme control. Cluc activity was simultaneously monitored to control for dose and transfection efficiencies. As shown, the catalytic activity of the previously described Schistosoma mansoni HHR variant, N107, was improved from its original 18-fold to 1,200-fold (blue bars) through a series of modifications, as represented above the figure. These include conversion of the type I HHR N107 to a type III HHR, optimization of stem III of the resulting type III ribozymes, modification of stem I to stabilize the tertiary interactions essential for ribozyme function, and alteration of loop I to facilitate hairpin formation. Data shown are representative of three independent experiments with similar results, and data points represent mean ± s.d. of three cell cultures. (c) Sequences and secondary structures of a natural Schistosoma mansoni HHR, the N107 ribozyme, and milestone type III ribozyme variants characterized in panel b. Differences between the native Schistosoma mansoni ribozyme and N107 are indicated in black. Successive modifications from N107 are indicated in blue. Further details are provided in the legend of Supplementary Figure 1.

Figure 2.. Efficient regulation of gene expression using optimized type III ribozymes.

(a) A diagram showing how an antisense oligonucleotide can inactivate a ribozyme to rescue protein expression. (b) The sequence and secondary structure of T3H16 ribozyme and the target regions complementary to the panel of phosphorodiamidate morpholino oligomers characterized in panel c. (c) 293T or Huh7 cells transfected with catalytically active or inactive T3H16 ribozyme-regulated reporter plasmids were treated with 10 μM of the indicated morpholino variants (M2 through M6) or an identical length control morpholino (NC). A functional morpholino’s activity was calculated as fold induction in the Gluc reporter expression relative to the Gluc expression from the NC control morpholino-treated cells. (d) Experiments similar to those in panel c except that the indicated cells were treated with an octa-guanidine dendrimer-coupled form of the M3 oligo, v-M3, and a similarly coupled control oligo, v-NC. (e) v-M3 morpholino-mediated induction of Gluc was compared using reporter plasmids with the T3H16 ribozyme placed at 3’ UTR or 5’ UTR. v-M3R is randomized from the v-M3 sequence. Note that induction is significantly (two-sample t-test, one-sided, P = 0.007) greater when the ribozyme is placed at 3’ UTR. (f) The sequences and secondary structures of three ribozyme variants, which have greater catalytic activity than T3H16 (T3H38, T3H48) or larger stem-I loop for morpholino targeting (T3H52), and the complementary target regions of the indicated morpholinos. (g) Octa-guanidine dendrimer-coupled forms of morpholinos M3 and M7-M10 (v-M3, v-M7 through v-M10) were tested in 293T and Huh7 cells for induction of Gluc expression from the corresponding ribozyme variant-controlled reporter plasmid. Numbers above the figure indicate the fold induction mediated by each morpholino. Data shown are representative of two or three independent experiments with similar results, and data points represent mean ± s.d. of three cell cultures.

Figure 3.. In vivo induction of an adeno-associated virus (AAV) reporter transgene.

(a) A diagram representing the AAV vector and the experimental design used in the animal studies of panels b through e. (b) Six-week old male BALB/c mice were intramuscularly (i.m.) injected in the left gastrocnemius muscle with 1×10¹⁰ genome copies (GC) of AAV particles carrying a T3H38-regulated firefly luciferase (Fluc) gene. Two weeks post AAV injection, mice were i.m. injected at the same site with 0.5 or 2.5 mg/kg of a control (v-NC) or the v-M8 morpholino. Bioluminescence imaging was performed at days 0 and 2 post-morpholino injection. These experiments were independently repeated three times with similar results. (c) Quantitation of luciferase expression shown in panel b. (d) An experiment similar to that shown in panel b except that 8-week old female BALB/c mice were i.m. injected at both hindlimbs with 5×10⁹ GC of T3H38-regulated AAV-Fluc. One group of mice then received an i.m. injection of 0.5 mg/kg v-M8 morpholino to one hindlimb and PBS to the other hindlimb. The other group received 12.5 mg/kg v-M8 morpholino via tail vein injection. Quantification of bioluminescence for the indicated time points is shown. Representative bioluminescent images for basal expression and induced peak expression are shown. (e) An experiment similar to that shown in panel b except that two groups of 8-week old female BALB/c mice were injected multiple times with the v-NC or v-M8 morpholino as indicated by the arrows. Bioluminescence imaging was performed at the indicated time points. Note that the third injection of group 2 was omitted due to a technical error. Numbers beside each peak indicate fold-induction over background measured before the first morpholino dose. Data points in panels c, d, and e represent mean ± s.d. of three, five, and four mice per group, respectively.

Figure 4.. In vivo regulation of an erythropoietin (Epo) transgene.

(a) A diagram representing the AAV vector and the experimental design used in the following animal studies. (b) Eight-week old female BALB/c mice were i.m. injected with 1×10¹⁰ GC of AAV particles carrying an active (T3H38a; upper right panel) or inactive (T3H38in; lower left panel) T3H38-regulated mouse erythropoietin (Epo) gene. Mice injected with active ribozyme-regulated AAV were further treated with 0.5 mg/kg of the v-M8 morpholino. Mice received no treatment (upper left panel) and a group of mice intraperitoneally injected with 3 μg recombinant mouse Epo protein (lower right panel) were used as controls. Hematocrit counts (blue lines) and plasma Epo protein concentrations (red lines) were measured at the indicated time points. Each line represents values obtained from a single mouse. The morpholino induction experiments were independently repeated twice with similar results. (c-f) Experiments similar to panel b except that the mice were injected with 5×10⁹ GC (c and d) or 2×10⁹ GC (e and f) of AAV particles carrying active T3H38-regulated mouse Epo gene and then treated with the indicated doses of the v-M8 morpholino. All differences among sets of mice treated with different morpholino concentrations are significant (paired-sample Student’s t-test, one-sided, P < 0.01 for panels c, e, and f, P < 0.05 for panel d) except for hematocrit values at 0.5 and 2.5 mg/kg (panel d). (g-h) Morpholino-induced peak Epo expression data from panel c are plotted by morpholino dose (g), and data from panels b, c, and e are plotted by AAV titers (h). Endogenous Epo concentrations (150 pg/ml) have been subtracted to determine fold induction of AAV-expressed Epo. The half-life of Epo induction and induction fold are shown above the figures Data points in panels c-h represent mean ± s.d. for three mice per group.