New therapeutic strategy for Huntington’s disease – HuntToTreat
Finding drugs and treatments that will alleviate the disease and/or its symtoms.
Proof of concept of a protective effect of P42 in vivo
Our project was to develop a strategy to inhibit the toxic effect of the polyQ-hHtt mutant protein. The starting point was the identification of a 23 aa peptide (P42) in the wild Htt protein, able to save different phenotypes induced by polyQ-hHtt mutant protein, in HeLa cells or in the fly. The use of peptides at therapeutic ends presents advantages (such as low toxicity), but also inconvenients. Indeed, peptides degrade quickly and have low reliability to reach the brain. To overcome these problems, we fused P42 to the transduction domain TAT (HIV), allowing its dissemination and addressing to the brain. In order to deliver P42 repetitively, using oral administrations (therefore non-invasive), we inserted the fusion peptide P42-TAT into water-in-oil microemulsions, using the Aonys technology developed by Medesis Pharma company.
The proper targetting and the effect of P42-TAT were analyzed on different models: i) in HeLa cells transfected with mutant polyQ-Htt; ii) in drosophila by genetic crosses; iii) in wild-type and R6/2 mice using daily treatment of microemulsions containing P42-TAT. In the latter case, the targetting into the brain was identified by microscopy and by the analysis of P42-TATderivatives by MALDI. The protective effect of P42-TAT was analyzed in mouse model of the disease on different phenotypes such as the weight of the mice, but also on their motor performances (by rotarod), reflex (testing foot clasping), but also post-mortem by analyzing the cerebral volume (using Nanozoomer) or by detecting biomarkers (counting aggregates or astrocytes).
These studies allowed the identification of a 23aa peptide (P42) quite effective against Huntington's disease, but also allowed the development of a technology for non-invasive administration (based on nanotechnology) that could be used for therapeutic purposes. These studies also allowed a proof of concept for asignificant benefit of P42 on Huntington's disease, which allowed its orphan drug designation by the European Medicines Agency. Our data also led to a collaboration with a group of Hong Kong, which has allowed us to obtain a new ANR to test P42 in multi therapies.
There is actually no drug that can delay the progression of this very devastating disease. Also the identification of a specific new product for this disease and thus with low toxicity is a major step forward. The peptidic therapies are made by injections often directly into the brain. Also the development of a non-invasive strategy using oral administration, and that we showed to be effective, may be used to deliver other molecules.
A patent is now recognized internationally. At European level, P42 was designated as an orphan drug. Two articles were published in 2013 and 2014: One on the identification of P42, the other on its action in mice.
Huntington’s disease (HD) is an inherited neurodegenerative disorder due to an abnormal expansion of a polyQ domain in the N-terminus of the human Huntingtin (hHtt) protein. When polyQ exceeds 35, polyQ-hHtt forms aggregates, and drives cell toxicity, leading to striatum neurodegeneration.
Although a number of molecular dysfunctions have been elucidated and help to explain the striatal neurodegeneration observed in HD, the exact mechanism whereby mutation in Huntingtin causes this neurodegeneration is still unclear. Several studies, including ours, indicate an influence of normal hHtt on the disease. Using a Drosophila model of HD, expressing a polyQ-hHtt, we could rescue polyQ-hHtt induced toxicity with the 548aa N-terminus part of hHtt or with the 620aa N-terminus part of Drosophila Ht (dHtt). These results suggest that HD must be considered as a dominant negative disease since the presence of expanded polyQ not only makes the mutated protein itself toxic, but also alters the normal functions of Htt. They also show that human and Drosophila Htt share biological properties, making Drosophila a privileged organism to study HD.
Therefore in a quest to find cures for HD, our project consists in developing a peptide-based strategy to protect cells against polyQ-hHtt toxicity. Based on our results indicating the protective role of normal Htt N-terminus, we recently identified a 23aa peptide (pep42) lying within the 548aa N-terminal part of the normal hHtt that is able to inhibit different phenotypes induced by the expression of the polyQ-hHtt in HeLa cells and in flies. Molecularly we identified how pep42 interferes with the first step of aggregation of the polyQ-hHtt (namely the nucleation step) through an interaction with the first N-terminal 17 aa (N17 region). At therapeutic ends, we now plan to fuse pep42 to a protein transduction domain such as (TAT-HIV) to be able to deliver the peptide into brain cells to treat the disease. The protective effect of the pep42-TAT fusion peptide will be therefore tested first in HeLa cells, then in entire organisms, notably in Drosophila, and in mice. The project benefits from the methodical characterization of the different neuronal defects that are induced by polyQ-hHtt in Drosophila that we have completed in our laboratory. This knowledge will be used to study the protective effect of pep42-TAT in vivo, comparative to pep42 alone, with the aim to also test their relative diffusion in an entire organism and their targeting in neurons. Finally tests will be performed in a mouse model of HD, where various delivery routes of pep42-TAT will be compared (intraventriculary or intravenous injections). Brain sections will be analyzed to test the efficiency of pep42-TAT delivery as well as its ability to rescue the polyQ-hHtt aggregates. Protective effects of the peptide will be also analyzed on mutated R6(2) mice associated behavioural defects. Once the proof of concept for the protective effect by pep42-TAT will be shown, an important issue will be to find more comfortable delivery process for the animal than injections, such as oral administration.
Madame Florence MASCHAT (Institut de Génomique Fonctionnelle - Eq Neurobiologie normale et pathologique chez la Drosophile ) – Florence.Maschat@igf.cnrs.fr
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
FIST France Innovation Scientifique et Transfert
IGF Institut de Génomique Fonctionnelle - Eq Neurobiologie normale et pathologique chez la Drosophile
Help of the ANR 250,175 euros
Beginning and duration of the scientific project: November 2012 - 24 Months