Increased brain bio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF

Abstract

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4+ T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.

Structure and biophysical characterization of the human full-length neurturin–GFRa2 complex: A role for heparan sulfate in signaling

“…Our results provide detailed molecular understanding of the HS binding site which has enabled us to rationally design point mutated NRTN variants, which should reduce the risk of structural perturbations or immunogenicity when used in vivo. These data suggest that NRTN variants with reduced affinity for HS could enable higher exposure upon injection or transgenic expression in target tissue (63). We have shown that point mutations in the HS binding site of NRTN can modify the affinity for HS and have confirmed that this translates to increased exposure and improved PK profile in vivo after both I.V.…”

Multiple signaling factors and drugs alleviate neuronal death induced by expression of human and zebrafish tau proteins in vivo

“…Glial cell-derived neurotrophic factor (GDNF) is another potent NT for a variety of neuronal populations [ 56 ]. Recently, it has been shown to have therapeutic potential for neurodegenerative disorders, including AD [ 57 ] and Parkinson disease (PD) [ 58 ].…”

“…4a and b ). In addition to the therapeutic potential for AD [ 57 ] and PD [ 58 ], this is the first report to indicate that GDNF has therapeutic potential for treating tau-induced neuronal death. In addition, chemical compounds have been shown to have the potential to stimulate synthesis and secretion of BDNF and GDNF in cultured astrocytes [ 71 ].…”

The Non-Survival Effects of Glial Cell Line-Derived Neurotrophic Factor on Neural Cells

“…GDNF signaling indisputably possesses potential therapeutic value for several pathologies, such as Parkinson’s disease or amyotrophic lateral sclerosis, among other diseases, where it has been tested using molecular and cellular approaches (Richardson et al, 2011 ; Thomsen et al, 2014 ; Rolan et al, 2015 ). In addition, the GDNF amino acid sequence has been engineered with modifications to improve its bio-distribution and half-life and to decrease its immunogenicity (Smith et al, 2015 ). The understanding of the entire range of properties of GDNF and other trophic molecules could provide opportunities to investigate their broad actions and accelerate and/or improve therapies based on these factors.…”