Amsterdam Molecular Therapeutics (AMT), a leader in the field of human gene therapy, announced the results from studies exploring the role of Adeno-Associated Viral (AAV) vectors for the efficient delivery of short and micro RNA to inhibit disease by RNA interference. Data related to the future development of an RNAi gene therapy for hypercholesterolemia and Huntington's disease were presented at the recent American Society of Gene and Cell Therapy 14th Annual Meeting in Seattle, USA.

“Our results demonstrate a robust long-term knock-down of ApoB by AAV-delivered miApoB in murine liver, thus providing an excellent candidate for the development of RNAi-based gene therapy,” stated Dr Harald Petry, Head of Research at AMT. “RNAi-based therapeutic strategies are considered highly promising in the industry, but thus far, effective delivery has been elusive. Our research has value in several therapeutic areas, such as hypercholesterolemia and associated cardiovascular disease. In addition, in Huntington's disease, progress is being made quickly through in vivo studies and we anticipate proof of concept data in animal models by the end of 2011 for this indication.”

Apolipoprotein B (ApoB) is a primary component of Low-Density Lipoprotein Cholesterol (LDL-C). Serum levels of LDL-C are considered to be proportionate to the risk of atherosclerotic vascular disease. AMT has conducted proof-of-concept studies in vivo, with AAV delivery of both short-hairpin RNA (AAV-shApoB) and microRNA (AAV-miApoB) to decrease ApoB gene expression, thereby successfully reducing cholesterol levels in serum.

AMT initially developed and screened 19 shRNAs targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed the focus to one candidate, shApoB10, for in vivo inhibition studies. To compare the intrinsic inhibitory properties and long-term efficacy of shRNA and miRNA, we expressed shApoB10 from a miRNA scaffold (miApoB) using the liver-specific LP1 promoter. Self-complementary Adeno-Associated Virus vector of serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB and miApoB.

In a comparative study, ApoB expression was significantly decreased in murine livers transduced with AAV-shApoB and AAV-miApoB. Expression of the AAV-shApoB and AAV-miApoB resulted in 90% ApoB protein knock-down, associated with 80% cholesterol decrease in murine plasma for the first 6 weeks. However, after 6 weeks the inhibitory effect of AAV-shApoB started to wear off, while AAV-miApoB retained a more stable inhibitory profile.

At 27 weeks post-injection, ApoB and plasma cholesterol were still decreased to 50% from baseline. Ongoing research at AMT aims to determine the mechanism underlying the differences seen between long-term AAV-shApoB and AAV-miApoB efficacy in murine livers using different AAV doses. The longer-term inhibitory effect of AAV-miApoB could be due to its lower toxicity and off-target properties compared to AAV-shApoB because expression of miApoB is specifically limited to hepatocytes.

One particularly promising focus of AMT in the development of artificial and cellular miRNAs targets several genes involved in HD, a rare and incurable neurodegenerative disease caused by poly-CAG expansions in the Huntington (Htt) gene. Initial studies have focused on optimizing the inhibitory RNA molecules to achieve highest allele specificity and efficacy against the mutant Htt gene. If the preliminary results are borne out by the proof of concept data, the HD programme would constitute an excellent program for a co-development agreement.

AMT is a world leader in the development of human gene based therapies. The company's lead product Glybera, a gene therapy for Lipo-Protein Lipase Deficiency (LPLD), is currently under review by the European Medicines Agency (EMA). If approved, Glybera will be the first gene therapy product to be marketed in Europe.