scholarly journals Investigation of a recombinant SMN protein delivery system to treat spinal muscular atrophy

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Ryan Anderton ◽  
Bruno Meloni ◽  
Frank Mastaglia ◽  
Sherif Boulos
Keyword(s):  
Spinal Muscular Atrophy ◽  
Cortical Neurons ◽  
Muscular Atrophy ◽  
Expression System ◽  
Cell Penetrating Peptides ◽  
Full Length ◽  
Dependent Manner ◽  
E Coli ◽  
Mammalian Expression ◽  
Smn Protein

AbstractSpinal muscular atrophy (SMA), the most common genetic cause of infant death, is a neurodegenerative disorder affecting motor neurons. SMA results from a loss in full-length survival of motor neuron (SMN) protein due to deletions/mutations in the SMN1 gene. In this study, we assessed the ability of cell-penetrating peptides (CPP) to deliver recombinant SMN protein to cultured neurons as a prelude for a potential therapeutic to treat SMA. Firstly, we confirmed that E. coli produced recombinant GFP protein fused to TAT (YGRKKRRQRRR; TAT-GFP) transduced rat cortical neurons in a concentration dependent manner. However, due to low yields of recombinant TATSMN protein obtainable from E. coli, we investigated the potential of a modified TAT (TATκ: YARKAARQARA) or R9 (RRRRRRRRR) peptide downstream of the fibronectin (FIB) secretory signal peptide to generate recombinant CPP-fused SMN protein. While U251 cells transduced with an adenoviral vector expressing CMV-FIB-TATκ-SMN secreted recombinant TATκ-SMN protein, we did not detect TATκ-SMN protein transduction of cortical neurons. Further, purified TATκ-SMN was unable to transduce SH-SY5Y cells, nor block apoptosis following LY294002 treatment of these cells. Our findings indicate that TATκ is not a suitable CPP to deliver SMN protein to neurons. Nonetheless, we have developed a novel method to generate full-length recombinant SMN protein using a mammalian expression system, which can be used to explore the application of other CPPs to deliver SMN protein as a treatment for SMA.

scholarly journals Recent Developments of Recombinant Protein Expression for Therapeutic Purposes

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Anam Amir
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Expression System ◽  
Cell Penetrating Peptides ◽  
Ovary Cell ◽  
Expression Systems ◽  
E Coli ◽  
Mammalian Expression ◽  
Mammalian Expression System ◽  
High Level

In the most recent seven to eight years, the therapeutic recombinant proteins have rapidly expanded in the biotechnology domain due to its wide variety of needs. There has been significant development in the mammalian expression system for fine purification and increased level of expressed recombinant proteins [1,2]. Many drugs like tetracycline have been demonstrated on the Chinese Hamster Ovary cell line for promising multi control strategies and effective cytotoxicity. Mammalian expression system improves the proper glycosylation of recombinant proteins which are very helpful to increase solubility of product [3-6].             Meanwhile on the prokaryotic expression system, E. coli has proven to be an easier to handle, friendly and economical strain [2]. Recently these expression systems are using to work on antibody fragment productions and their proper folding with co-expression of chaperones [7]. Moreover E. coli has been used for the production of cancer cell penetrating peptides which promises the targeted delivery of drugs to specific effector cells only.  Yeast systems are also being used for the antibody fragments production and the high level production of insulin. Interestingly cell free expression systems are also participating in this game and that would be very fascinating to see in the coming years about cell extract medium for production of high level recombinant protein [8, 9]. Purification and optimization of recombinant protein has always been a challenging situation for scientists and they paid more attention to increase the overall yield of the product. Many affinity chromatography techniques has been introduced for efficient purification of protein of interest [10]. Despite these research and developments in methodologies to produce and purify the recombinant therapeutic protein, scientists still face the hurdles and challenges with all expression systems. Rationally E. coli produces inclusion bodies and many mammalian cell types do not show the same results with the same recombinant protein. [11]. So there is a requirement for adding the appropriate features to the expression systems focused to better improvising recovery, production and purification of recombinant protein. Copyright(c) The Author

SMN mRNA and protein levels in peripheral blood

Neurology ◽  
2006 ◽  
Vol 66 (7) ◽  
pp. 1067-1073 ◽  
Author(s):  
C. J. Sumner ◽  
S. J. Kolb ◽  
G. G. Harmison ◽  
N. O. Jeffries ◽  
K. Schadt ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Spinal Muscular Atrophy ◽  
Disease Severity ◽  
Peripheral Blood ◽  
Muscular Atrophy ◽  
Gene Copy ◽  
Blood Levels ◽  
Type I ◽  
Protein Levels ◽  
Smn Protein

Background: Clinical trials of drugs that increase SMN protein levels in vitro are currently under way in patients with spinal muscular atrophy.Objective: To develop and validate measures of SMN mRNA and protein in peripheral blood and to establish baseline SMN levels in a cohort of controls, carriers, and patients of known genotype, which could be used to follow response to treatment.Methods: SMN1 and SMN2 gene copy numbers were determined in blood samples collected from 86 subjects. Quantitative reverse transcription PCR was used to measure blood levels of SMN mRNA with and without exon 7. A cell immunoassay was used to measure blood levels of SMN protein.Results: Blood levels of SMN mRNA and protein were measured with high reliability. There was little variation in SMN levels in individual subjects over a 5-week period. Levels of exon 7-containing SMN mRNA and SMN protein correlated with SMN1 and SMN2 gene copy number. With the exception of type I SMA, there was no correlation between SMN levels and disease severity.Conclusion: SMN mRNA and protein levels can be reliably measured in the peripheral blood and used during clinical trials in spinal muscular atrophy, but these levels do not necessarily predict disease severity.

scholarly journals Calpain Inhibition Increases SMN Protein in Spinal Cord Motoneurons and Ameliorates the Spinal Muscular Atrophy Phenotype in Mice

2018 ◽  
Vol 56 (6) ◽  
pp. 4414-4427 ◽  
Author(s):  
Sandra de la Fuente ◽  
Alba Sansa ◽  
Ambika Periyakaruppiah ◽  
Ana Garcera ◽  
Rosa M. Soler
Keyword(s):  
Spinal Cord ◽  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Smn Protein ◽  
Calpain Inhibition

scholarly journals Restoration of SMN to Emx-1 expressing cortical neurons is not sufficient to provide benefit to a severe mouse model of Spinal Muscular Atrophy

2013 ◽  
Vol 22 (5) ◽  
pp. 1029-1036 ◽  
Author(s):  
Alexander S. Taylor ◽  
Jacqueline J. Glascock ◽  
Ferrill F. Rose ◽  
Cathleen Lutz ◽  
Christian L. Lorson
Keyword(s):  
Mouse Model ◽  
Spinal Muscular Atrophy ◽  
Cortical Neurons ◽  
Muscular Atrophy

scholarly journals Molecular Mechanisms of Neurodegeneration in Spinal Muscular Atrophy

2016 ◽  
Vol 10 ◽  
pp. JEN.S33122 ◽  
Author(s):  
Saif Ahmad ◽  
Kanchan Bhatia ◽  
Annapoorna Kannan ◽  
Laxman Gangwani
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Muscular Atrophy ◽  
Survival Motor Neuron ◽  
Skeletal Muscle Atrophy ◽  
Spinal Motor Neurons ◽  
Low Levels ◽  
Smn Protein

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA.

scholarly journals Temporal and tissue-specific variability of SMN protein levels in mouse models of spinal muscular atrophy

2018 ◽  
Vol 27 (16) ◽  
pp. 2851-2862 ◽  
Author(s):  
Ewout J N Groen ◽  
Elena Perenthaler ◽  
Natalie L Courtney ◽  
Crispin Y Jordan ◽  
Hannah K Shorrock ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Mouse Models ◽  
Muscular Atrophy ◽  
Tissue Specific ◽  
Protein Levels ◽  
Smn Protein

scholarly journals Muscle overexpression of Klf15 via an AAV8-Spc5-12 construct does not provide benefits in spinal muscular atrophy mice

Gene Therapy ◽  
2020 ◽  
Vol 27 (10-11) ◽  
pp. 505-515
Author(s):  
Nina Ahlskog ◽  
Daniel Hayler ◽  
Anja Krueger ◽  
Sabrina Kubinski ◽  
Peter Claus ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Treatment Options ◽  
Survival Motor Neuron ◽  
Significant Activity ◽  
Dependent Manner ◽  
Patient Response ◽  
High Gene Expression ◽  
Virus Serotype ◽  
High Gene

AbstractSpinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the survival motor neuron (SMN) gene. While there are currently two approved gene-based therapies for SMA, availability, high cost, and differences in patient response indicate that alternative treatment options are needed. Optimal therapeutic strategies will likely be a combination of SMN-dependent and -independent treatments aimed at alleviating symptoms in the central nervous system and peripheral muscles. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates key metabolic and ergogenic pathways in muscle. We have recently reported significant downregulation of Klf15 in muscle of presymptomatic SMA mice. Importantly, perinatal upregulation of Klf15 via transgenic and pharmacological methods resulted in improved disease phenotypes in SMA mice, including weight and survival. In the current study, we designed an adeno-associated virus serotype 8 (AAV8) vector to overexpress a codon-optimized Klf15 cDNA under the muscle-specific Spc5-12 promoter (AAV8-Klf15). Administration of AAV8-Klf15 to severe Taiwanese Smn−/−;SMN2 or intermediate Smn2B/− SMA mice significantly increased Klf15 expression in muscle. We also observed significant activity of the AAV8-Klf15 vector in liver and heart. AAV8-mediated Klf15 overexpression moderately improved survival in the Smn2B/− model but not in the Taiwanese mice. An inability to specifically induce Klf15 expression at physiological levels in a time- and tissue-dependent manner may have contributed to this limited efficacy. Thus, our work demonstrates that an AAV8-Spc5-12 vector induces high gene expression as early as P2 in several tissues including muscle, heart, and liver, but highlights the challenges of achieving meaningful vector-mediated transgene expression of Klf15.

scholarly journals The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

2005 ◽  
Vol 25 (13) ◽  
pp. 5543-5551 ◽  
Author(s):  
Lili Wan ◽  
Daniel J. Battle ◽  
Jeongsik Yong ◽  
Amelie K. Gubitz ◽  
Stephen J. Kolb ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neurons ◽  
Muscular Atrophy ◽  
Protein Levels ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Smn Complex ◽  
Survival Of Motor Neurons ◽  
Smn Protein ◽  
Nuclear Ribonucleoprotein

ABSTRACT Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

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