Summary Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here we tested this hypothesis using AR113Q knock-in and human BAC transgenic mice that express the full-length polyQ AR and display androgen-dependent weakness, muscle atrophy and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery, but not CNS, following subcutaneous administration. Suppression of polyQ AR in the periphery rescued deficits in muscle weight, fiber size and grip strength, reversed changes in muscle gene expression, and extended lifespan of mutant males. We conclude that polyQ AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

Objective Nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of advanced chronic liver disease. The progression of NAFLD, including nonalcoholic steatohepatitis (NASH), has a strong genetic component, and the most robust contributor is the patatin-like phospholipase domain-containing 3 ( PNPLA3 ) rs738409 encoding the 148M protein sequence variant. We hypothesized that suppressing the expression of the PNPLA3 148M mutant protein would exert a beneficial effect on the entire spectrum of NAFLD. Methods We examined the effects of liver-targeted GalNAc 3 -conjugated antisense oligonucleotide (ASO)-mediated silencing of Pnpla3 in a knock-in mouse model in which we introduced the human PNPLA3 I148M mutation. Results ASO-mediated silencing of Pnpla3 reduced liver steatosis ( p = 0.038) in homozygous Pnpla3 148M/M knock-in mutant mice but not in wild-type littermates fed a steatogenic high-sucrose diet. In mice fed a NASH-inducing diet, ASO-mediated silencing of Pnpla3 reduced liver steatosis score and NAFLD activity score independent of the Pnpla3 genotype, while reductions in liver inflammation score ( p = 0.018) and fibrosis stage ( p = 0.031) were observed only in the Pnpla3 knock-in 148M/M mutant mice. These responses were accompanied by reduced liver levels of Mcp1 ( p = 0.026) and Timp2 ( p = 0.007) specifically in the mutant knock-in mice. This may reduce levels of chemokine attracting inflammatory cells and increase the collagenolytic activity during tissue regeneration. Conclusion This study provides the first evidence that a Pnpla3 ASO therapy can improve all features of NAFLD, including liver fibrosis, and suppress the expression of a strong innate genetic risk factor, Pnpla3 148M, which may open up a precision medicine approach in NASH.

Advances in the medicinal chemistry of antisense oligonucleotide drugs have been instrumental in achieving and optimizing antisense activity in cell types other than hepatocytes, the cell type that is most sensitive to antisense effects following systemic treatment. To broadly characterize the effects of antisense drugs on target messenger RNA (mRNA) levels in different organs and cell types in animals, we have developed a sensitive RNA in situ hybridization technique using the noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) as a surrogate target. We have used this technique to evaluate the effects of 2'-O-methoxy ethyl (MOE) and constrained ethyl bicyclic nucleic acid (cEt) gapmer antisense oligonucleotides (ASOs). ASO tissue distribution was also characterized using immunohistochemical techniques, and MALAT1 mRNA reductions were confirmed by quantitative real time-polymerase chain reaction. Our findings demonstrate that systemic antisense drug administration in both mice and non-human primates resulted in marked reductions in MALAT1 RNA in many tissues and cell types other than liver including kidney, muscle, lung, adipose, adrenal gland, and peripheral nerve tissue. As expected, ASOs with cEt chemistry were more efficacious than MOE ASO in all tissues examined.

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

The scarring response is an important factor in many diseases throughout the body. In addition, it is a major problem in influencing results of surgery. In the eye, for example, post-operative scarring can determine the outcome of surgery. This is particularly the case in the blinding disease glaucoma, where several anti-scarring regimens are currently used to improve glaucoma surgery results, but are of limited use clinically because of severe complications. We have recently identified transforming growth factor-b (TGF-b) as a target for post-operative anti-scarring therapy in glaucoma, and now report the first study of novel secondgeneration antisense phosphorothioate oligonucleotides against TGF-b in vivo. Single applications of a TGF-b OGN at the time of surgery in two different animal models closely related to the surgical procedure performed in glaucoma patients, significantly reduced post-operative scarring (Po0.05) and improved surgical outcome. Our findings suggest that TGF-b antisense oligonucleotides have potential as a new therapy for reducing post-surgical scarring. Its long-lasting effects after only a single administration at the time of surgery make it particularly attractive clinically. Furthermore, although we have shown this agent to be useful in the eye, it could have widespread applications anywhere in the body where the wound-healing response requires modulation.