Glial cell line-derived neurotrophic factor: Characterization of mammalian posttranslational modifications

Piccinini, Elisa; Kalkkinen, Nisse; Saarma, Märt; Runeberg-Roos, Pia

doi:10.3109/07853890.2012.663927

Cited by 26 publications

(24 citation statements)

References 47 publications

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“…However, we do not anticipate that these changes will increase the proteolytic stability in vivo and hence this parameter was not measured in our experiments. .GDNFv is expressed in CHO cells and is glycosylated at one position N49 (data not shown), N49 mutations to remove glycosylation in mammalian expressed GDNF resulted in significant lower expression levels, consistent with the finding by Piccinini et al (2013) . It was also observed that full length unglycosylated E. coli produced GDNFwt has significantly higher hydrophobicity than GDNFv produced by mammalian expression (data not shown).…”

Section: Discussionsupporting

confidence: 87%

“…As result of bacterial expression, the molecule has an additional N-terminal methionine and is unglycoslyated. Piccinini et al (2013) recently showed that GDNFwt purified from E. coli had poor stability compared to mammalian expressed GDNF. Since the drug may need to reside in a pump/catheter system at body temperature for many weeks to months during infusion to PD patients, we have conducted stability studies with GDNFwt and identified two major deamidation and isomerization sites, Asn38 and Asp95.…”

Section: Discussionmentioning

confidence: 99%

“…This observation raises concerns regarding the immunogenicity of GDNFwt, which potentially could impact both efficacy and safety of the drug, although no adverse consequences were reported in the trial (Tatarewicz et al, 2007). Finally, Piccinini et al (2013) recently showed that GDNFwt expressed and purified from E. coli had poor stability, raising this as another limitation to GDNFwt as a therapeutic for PD, since the drug may need to reside in a pump/catheter system at body temperature for many weeks to months prior to infusion.…”

Section: Introductionmentioning

confidence: 93%

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

Smith

O’Bryan

Mitchell

et al. 2015

Experimental Neurology

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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.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 93%

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

Smith

O’Bryan

Mitchell

et al. 2015

Experimental Neurology

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“…The production method can also be important for the activity and solubility of the protein. For example, GDNF produced in E.coli is non-glycosylated whereas GDNF produced in mammalian cells is glycosylated and more stable 70 . The advantages of protein delivery are that protein receptor mediated signaling is limited by its half-life and the administration techniques via pumps can be intermittent and easily terminated.…”

Section: Gdnf-and Cdnf/manf-families Of Neurotrophic Factors: Effectsmentioning

confidence: 99%

Prospects of Neurotrophic Factors for Parkinson's Disease: Comparison of Protein and Gene Therapy

2015

Self Cite

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Neurotrophic factors (NTFs) hold great potential as therapeutic agents in the treatment of neurodegenerative conditions, including Parkinson's disease (PD), in which the progressive loss of dopamine neurons in the substantia nigra pars compacta causes severe motor symptoms. There is extensive evidence that in preclinical animal models of PD NTFs are both neuroprotective and neurorestorative. In particular, glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), cerebral dopamine neurotrophic factor, and mesencephalic astrocyte-derived neurotrophic factor have shown great potential to restore dopamine neurocircuitry. Although some previous clinical trials have demonstrated limited efficacy of GDNF and NRTN, there are several concerns raised with these studies. Moreover, open-label studies with GDNF as well as a study with NRTN showed clinical improvement, particularly in patients with early-stage PD. Indeed, as previous clinical trials with NTFs were associated with several technical problems, there is a great need for further investigations. In this review we discuss the emerging and existing possibilities to use NTFs as neurorestorative agents and the ways to improve their efficacy, and compare gene therapy and recombinant protein therapy approaches for restoring the dopamine circuitry in PD.

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“…As a multifunctional protein, GDNF has the ability to induce cellular survival, proliferation, migration and differentiation (13). To date, the scope of GDNF applications has been greatly expanded and includes the treatment of Huntington’s disease, amyotrophic lateral sclerosis, chronic pain, depression and addiction, as well as the regeneration of the sciatic nerve (14). In view of its numerous applications, GDNF was selected as the target protein in the present study.…”

Section: Discussionmentioning

confidence: 99%

Feasibility of using a dual-promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells

Wang

Cai

et al. 2014

Experimental and Therapeutic Medicine

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Vectors that are capable of coexpressing two or more exogenous genes for in vitro and in vivo gene delivery are being increasingly studied. The aim of the present study was to explore the feasibility of using the pFastBac™ Dual vector, under the control of two cytomegalovirus (CMV) promoters with opposite directions, to coexpress enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) in the same mammalian cell. In the study, two promoters in the pFastBac Dual vector were replaced with CMV-EGFP and CMV-GDNF, whose directions were consistent with the initial directions. The pFastBac Dual-CMV-EGFP-CMV-GDNF plasmid was constructed and then transfected into human embryonic kidney (HEK) 293T cells. The recombinant virus, Bac Dual-CMV-EGFP-CMV-GDNF, was generated with the Bac-to-Bac Baculovirus Expression system and used to transduce HeLa cells. Immunofluorescence was applied to examine the coexpression of EGFP and GDNF in transfected or transduced mammalian cells, while western blot analysis was used to confirm the expression of GDNF in transduced HeLa cells. The recombinant plasmid was constructed and the recombinant baculovirus was successfully generated. Immunofluorescence observations demonstrated that EGFP and GDNF were simultaneously expressed in the same transfected HEK 293T cell and in a single transduced HeLa cell. Western blot analysis revealed that GDNF was expressed accurately in the transduced cells. Therefore, the pFastBac Dual vector is an efficient gene transfer vector that is able to coexpress two target proteins in mammalian cells and serve as a platform for combining reporter or/and therapy genes used in molecular imaging and dual-gene therapy. Thus, the current study presents a new coexpression strategy for dual-gene delivery in vitro and in vivo.

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Glial cell line-derived neurotrophic factor: Characterization of mammalian posttranslational modifications

Abstract: Posttranslational modifications of GDNF influence its stability, which may be critical for its clinical use.

Cited by 26 publications

References 47 publications

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

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

Prospects of Neurotrophic Factors for Parkinson's Disease: Comparison of Protein and Gene Therapy

Feasibility of using a dual-promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells

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