Engineering viral vectors for acoustically targeted gene delivery

Abstract

Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, which limit its applicable scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency and results in substantial undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity in two tested mouse strains. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms.

Fig. 1. Screening methodology for generation of an AAV for improved site-specific noninvasive gene delivery to the brain.

a Summary of the high-throughput screening and selection process. AAV library is administered intravenously (I.V.) and delivered to one brain hemisphere through FUS-BBBO. After 14 days mice are euthanized, their brain harvested, and the DNA from selected hemispheres is extracted. The DNA is then amplified by Cre-dependent PCR that enriches the viral DNA modified by Cre. In our case, neurons expressed Cre exclusively, and the Cre-dependent PCR enriched viral DNA of AAVs that transduced neurons. We subjected the obtained viral DNA to next-generation sequencing for the targeted hemisphere (round 1) or both targeted and control hemispheres (round 2). The process is then repeated for the next round (steps exclusive to round 2 indicated by the gray text). b Overall, 1.3 billion clones were screened in the first round, and 2098 clones in the second round of selection. Out of these clones, we selected 5 that were tested in low-throughput to yield AAV.FUS.3—a vector with enhanced FUS-BBBO gene delivery.

Fig. 2. High throughput screening yields vectors with improved FUS-BBBO gene delivery.

a An MRI image showing mouse brain with 4 sites opened with FUS-BBBO in one hemisphere. The bright areas (arrowheads) indicate successful BBB opening and extravasation of the MRI contrast agent Prohance into the brain. This BBB opening was used for delivery of the AAV library. b Sequencing results of round 2 of screening show a fraction of NGS reads within the DNA extracted from brains of Syn1-Cre mice subjected to FUS-BBBO and injected with a focused library of 2098 clones. Each dot represents a unique capsid protein sequence, and the position on each axis corresponds to the number of times the sequence was detected in the FUS-targeted and untargeted hemispheres. Markers below the dotted line represent sequences that on average showed 100-fold higher enrichment in the targeted hemisphere as compared to the control hemisphere. Dark gray dots represent 22 clones that are enriched in the FUS targeted hemispheres at least 100-fold in every tested mouse and DNA sequence encoding the 7-mer insertion peptide. Additional 13 clones had zero detected transduction in the untargeted hemisphere and could not be presented on the log-log plot. Yellow dots represent 5 clones (AAV.FUS.1-5) selected for low-throughput testing. Due to the use of a logarithmic plot, clones that had zero copies detected in either of the hemispheres are not shown. Data from one male and one female mouse.

Fig. 3. AAV.FUS candidates improve efficiency of gene delivery to the brain and reduce peripheral transduction.

a Representative images were obtained from mice co-injected with AAV9 and a AAV.FUS.3 at 10¹⁰ viral genomes per gram of body weight each. Sections show brain transduction by AAV9 (red) and AAV.FUS.3 (green), and are counterstained with a neuronal stain (NeuN, blue). b All but one (AAV.FUS.4) AAV.FUS candidates showed significant improvement over the co-injected AAV9. (p-values for AAV.FUS.1-5, p = 0.0274, 0.0003, 0.0052, 0.2556, 0.0087, respectively; Two-way ANOVA with Sidak multiple comparisons test: F(1, 24) = 59.49, P value; P < 0.0001). Data from 3 male and 3 female mice per serotype. c We found that few cells were transduced outside of the FUS-targeted site and AAV.FUS.3 and AAV9 were not significantly different. (0.19% vs 0.4%, respectively; p = 0.072, two-way ANOVA with Sidak multiple comparisons test; F(1, 35) = 2.457, p = 0.1260). Similarly, other candidates also showed no differences compared to AAV9 (AAV.FUS.1, p = 0.99; AAV.FUS.2, p = 0.98; AAV.FUS.4, p = 0.86; AAV.FUS.5, p = 0.83). Data from 3 male and 3 female mice for all serotypes, except AAV.FUS.2 (2 male, 3 female mice), and AAV.FUS.3 (8 male and 8 female mice). d Representative images showing liver transduction by AAV9 (red) and AAV.FUS.3 (green). e All tested candidates showed reduced liver transduction as compared to the co-injected AAV9 in the same mice for which brain expression was analyzed. (P-values for AAV9 vs AAV.FUS.1-5 were p = 0.0058 for AAV.FUS.1, and p < 0.0001 for other candidates; Two-way ANOVA, F(1, 24) = 375.9, P < 0.0001. Data from 3 male and 3 female mice for all serotypes, except AAV.FUS.2 (2 male, 3 female mice). f We defined the fold-improvement in targeting efficiency as the ratio of brain transduction to the liver transduction efficiency using AAV9 as a baseline, which suggested that AAV.FUS.3 is the top candidate for further study. (AAV.FUS.3 compared to AAV.FUS.1,2,4,5, all p-values were p < 0.0001, one way ANOVA with Tukey HSD post hoc comparison test). Scale bars are 50 μm in (a, c), unless otherwise noted. (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05, ns = not significant); Error bars are 95% CI. The numbers of animals used in each experiment were: Data from 3 male and 3 female mice per serotype, except AAV.FUS.2 (2 male, 3 female mice). Center for the error bars represents arithmetic mean in (b, c, e, f).

Fig. 4. AAV.FUS candidates show improved neuronal tropism.

a All AAV.FUS candidates show improved neuronal tropism upon FUS-BBBO gene delivery. AAV.FUS.3 had 56% more likelihood of transducing a neuron than AAV9 (69.8%, vs 44.7% neuronal transduction, respectively; (for all samples p < 0.0001, one way ANOVA, F(5, 31) = 52.60, P < 0.0001; n = 8 for AAV9, n = 6 for all AAV.FUS.1,3,4,5, n = 5 for AAV.FUS.2, center for the error bars represents arithmetic mean.). b Representative images showing AAV9 transducing both neurons (blue, NeuN staining, example neurons designated by an arrow) and non-neuronal cells (example non-neuronal cells designated by an arrowhead). c In comparison, more of the cells transduced with AAV.FUS (green) are neurons (example neurons designated by an arrow), rather than non-neuronal cells (example cell designated by an arrowhead). IV injection dose, 10¹⁰ vg/g. Scale bars are 50 μm. (****p < 0.0001). Error bars are 95% CI.

Fig. 5. AAV.FUS.3 shows regional dependence of transduction efficiency.

Hippocampus showed the highest, 4.3-fold, improvement in transduction over AAV9. a Representative image comparing transduction of the cortex with AAV.FUS.3 (green) and AAV9 (red). b Representative image comparing transduction of the striatum with AAV.FUS.3 (green) and AAV9 (red). c Representative image comparing transduction of the thalamus with AAV.FUS.3 (green) and AAV9 (red). d Representative image comparing transduction of the hippocampus with AAV.FUS.3 (green) and AAV9 (red). e Representative image comparing transduction of the midbrain with AAV.FUS.3 (green) and AAV9 (red). f AAV.FUS.3 shows regional differences in transduction efficiency of the tested regions – cortex (Ctx), striatum (Str), thalamus (Th), hippocampus (Hpc), midbrain (Mb). All differences were statistically significant (All pairwise comparison p-values < 0.0001, except thalamus vs striatum (p = 0.0026) and striatum vs midbrain (p = 0.0015), n = 3 mice per region, one way ANOVA, F(4, 10) = 283.4, P < 0.0001; Tukey HSD post-hoc test; center for the error bars represents arithmetic mean.). g Neuronal transduction efficiency for AAV9 (gray) and AAV.FUS.3 (orange). AAV.FUS.3 showed significant improvement over AAV9 transduction in all tested regions, n = 3 mice per region (two-way ANOVA with Sidak’s test; F(1, 20) = 141.2; p = 0.0333, p < 0.0001, p = 0.0002, p < 0.0001, p < 0.001 for Cortex, Striatum, Thalamus, Hippocampus and Midbrain, respectively; center for the error bars represents arithmetic mean.). IV injection dose, 10¹⁰ VG/g. Scale bars are 50 μm. Error bars are 95% CI.