Huntington's disease (HD) is a devasting, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene. Inactivation of the mutant allele by CRISPR-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mutant HTT (mHTT) protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in serum-deprived conditions are enriched in six protein pathways and one lipid class, dilysocardiolipin. Inspired by these findings, we engineer an "artificial exosome," in which the incorporation of one lipid (dilysocardiolipin) and three proteins (Rab7, Desmoplakin, and AHSG) into conventional neutral liposomes produces vesicles that mimic cargo delivering activity of natural exosomes.

Exosomes can deliver therapeutic RNAs to neurons. The composition and the safety profile of exosomes depend on the type of the exosome-producing cell. Mesenchymal stem cells are considered to be an attractive cell type for therapeutic exosome production. However, scalable methods to isolate and manufacture exosomes from mesenchymal stem cells are lacking, a limitation to the clinical translation of exosome technology. We evaluate mesenchymal stem cells from different sources and find that umbilical cord-derived mesenchymal stem cells produce the highest exosome yield. To optimize exosome production, we cultivate umbilical cord-derived mesenchymal stem cells in scalable microcarrier-based three-dimensional (3D) cultures. In combination with the conventional differential ultracentrifugation, 3D culture yields 20-fold more exosomes (3D-UC-exosomes) than two-dimensional cultures (2D-UC-exosomes). Tangential flow filtration (TFF) in combination with 3D mesenchymal stem cell cultures further improves the yield of exosomes (3D-TFF-exosomes) 7-fold over 3D-UC-exosomes. 3D-TFF-exosomes are seven times more potent in small interfering RNA (siRNA) transfer to neurons compared with 2D-UC-exosomes. Microcarrier-based 3D culture and TFF allow scalable production of biologically active exosomes from mesenchymal stem cells. These findings lift a major roadblock for the clinical utility of mesenchymal stem cell exosomes.

Small extracellular vesicles (sEVs) show promise as natural nano-devices for delivery of therapeutic RNA, but efficient loading of therapeutic RNA remains a challenge. We have recently shown that the attachment of cholesterol to small interfering RNAs (siRNAs) enables efficient and productive loading into sEVs. Here, we systematically explore the ability of lipid conjugates-fatty acids, sterols, and vitamins-to load siRNAs into sEVs and support gene silencing in primary neurons. Hydrophobicity of the conjugated siRNAs defined loading efficiency and the silencing activity of siRNA-sEVs complexes. Vitamin-E-conjugated siRNA supported the best loading into sEVs and productive RNA delivery to neurons.

Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5'-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5'-(E)-vinylphosphonate and alternating 2'-fluoro and 2'-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at approximately 3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles.

Huntington's disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken beta-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CbetaA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CbetaA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CbetaA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CbetaA promoter can provide an effective and safe dose of a human huntingtin miRNA.