Refining the bioactivity of P42, a hit therapeutic peptide, and developing a combined therapeutic peptide approach for treating Huntington’s Disease. – PEP-FOR-HD

Peptides have potentially a low toxicity, but they normally hardly cross the blood brain barrier and they are rapidly degraded. One issue is to develop peptides with the best stability and bioactivity. Whereas we already identified an active form of P42, efforts are made to enhance its cell penetrating ability. We have also constructed fusion P42 peptides that may enhance the degradation of the mutated polyQ-Htt. Finally HYE Chan recently identified modifications that enhance the stability of the P3 peptide. We may apply such modifications on P42.
In parallele we developed different tests in cellulo to compare the activity of different sets of P42 modified peptides and select the most active peptide in order to test these peptides in vivo on R6/2 model of HD..

The challenge was to better characterize P42 and P3, in order to perform a P42/P3 combined therapy. Concerning P3, its identification has been published in 2016 by the group of Edwin Chan from Hong Kong, who is our project partner (Zhang et al., Disease Models & Mechanisms, 2016). As part of Huntingtin protein, the physiological role of P42 has been studied. Interestingly, we identified that P42 is acting at different levels of the disease, not only on the aggregation process as previously described (Marelli et Maschat, Orphanet Journal of Rare Diseases, 2016), but also on BDNF signalling pathway, which is impacting associated phenotypes such as anxiety, memory or locomotion (Article submitted).
These data suggest that P42 may be protective of Huntington's disease, not only by reducing the negative effect of polyQ-Htt mutant protein through the inhibition of the aggregates formation, but also by increasing the activity of normal Huntingtin. This provides a particularly interesting therapeutic potential for P42, in the context of this disease.
As proposed in this project, we also investigated P3 activity in mice. This allowed to identify that P3 is able to enhance neuronal activity, and a higher activity of the mice.
Using P42/P3 bitherapy test on R6/2 mice showed that globally the recovery is more efficient than P3, but stays close to P42, even though with more robust recovery.

Our focus is to improve the bioactivity of our peptides and identify the most efficient way of administration for the bitherapy.
Our results already suggested that P3 is active in particular in locomotion. Flies expressing P3 that we constructed will be tested accordingly.
Our results also showed that P3 activity requires investigating for a better stability and bioactivity. This part has been pursued by HYE Chan’s laboratory (Article submitted). The activity of this modified P3 will be tested on Ca2+ activity on hippocampal cells.
HYE Chan is also exploring other ways of administration of the peptides. We will be able to compare bitherapies using either Aonys® technology to these new methods.

An article about the protective effect of P42 in a mouse model of Huntington's disease was published in 2014 (Arribat et al, (2014) Systemic delivery of P42 peptide: A new weapon to fight Huntington's disease, Acta Neuropathologica Communications , 2:86).
Additionally to our recent data, this allowed us to achieve in 2015 an Orphan Drug designation by the EMA (European Medicines Agency WC500188876), acclaimed by the CNRS (innovation letter and vimeo vimeo.com/152021281) and by Inserm. This study led to the publication of a review (Marelli, C., Maschat, F. (2016), P42: A novel and promising peptide-based therapy for Huntington's disease., Orphanet Journal of Rare Diseases 11:24).
Patent PCT/FR2012/050809, 04/12/2012 were obtained at the international (US and Australia), and are pending (Europe and Canada).
Importantly we have investigated the possibility to use P42 not only to prevent but also to treat HD. The identification of P42 effect on BDNF signaling when provided at different phases of the disease confirmed this possibility (article submitted: Improvement of BDNF signalling by P42 peptide in Huntington's disease. A. Paucard#, S. Couly#, (# equal contribution), N. Bonneaud, T. Maurice, L. Benigno, C. Jourdan, M. Vignes and F. Maschat.).

Huntington’s Disease (HD) is caused by the genomic expansion of glutamine-encoding CAG triplet repeats in the coding region of the IT15 locus in the human genome, which encodes the Huntingtin (hHtt) protein. It is now generally accepted that hHtt protein toxicity originates from protein misfolding and aggregation. Misfolded/aggregated hHtt triggers deleterious events in neurons, which can result in neuronal dysfunction and cell death. In 2013, F. Maschat’s team identified a 23 aa peptide, P42, that possesses the ability to 1) reduce protein aggregation of the HD disease protein, mutant Huntingtin (polyQ-hHtt), 2) rescue HD neuronal phenotypes including axonal transport and locomotor defects, and 3) extend mean survival time of HD transgenic flies in vivo. Consistent protective effects were also observed in HD transgenic mice. In addition to protein toxicity, it has been reported that mutant polyQ disease mRNA transcripts also contribute to neurotoxicity in polyQ degeneration, including HD. E. Chan’s team reported the role of RNA toxicity in HD pathogenesis, and developed a 13 aa CAG-RNA toxicity peptidylic inhibitor, P3, which possesses the activity to suppress 1) pre-45s rRNA transcription defects, 2) nucleolar stress induction and 3) neurodegeneration caused by CAG-RNA toxicity in vivo. Since both expanded polyQ-hHtt protein and hHtt CAG-RNA contribute to HD pathogenesis, a therapeutic strategy that simultaneously targets both of these toxic species would be ideal for treating HD. Our aim in the proposed work are (1) to optimize the bioactivities of peptidylic inhibitors using peptide engineering techniques; (2) to develop efficient peptide delivery systems to co-deliver the RNA and protein peptidylic inhibitors to the brain of HD disease mouse model; and (3) to optimize the therapeutic effect of RNA and protein toxicity co-treatment in HD mouse model. In the long run, our work will open up new therapeutic options for HD.