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

Abstract

Spinal 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.

AAV vectors: The Rubik’s cube of human gene therapy

“…Recently, Zolgensma, also known as AVXS-101, was approved by the FDA to treat SMA. SMA is caused by a mutation in the survival motor neuron 1 (SMN1) gene, 336 , 337 occurs in approximately 1 in 10,000 live births, and is the leading genetic cause of infant mortality. 338 , 339 This AAV9-based drug consists of double-stranded DNA and is used to treat pediatric patients less than 2 years of age with bi-allelic mutations in the SMN1 gene.…”

The Argi system: one-step purification of proteins tagged with arginine-rich cell-penetrating peptides

“…Although CPP-based therapy has not yet entered human, but only in vitro and animal clinical trials, the current results may be cautiously considered as optimistic 7 . There are numerous reports where successful transport of various cargos into different types of cells including plant cells using arginine-rich peptides, was demonstrated 8 – 14 .…”

Lack of Evidence for Decreased Protein Stability in the 2397 (Met) Haplotype of the Leucine Rich Repeat Kinase 2 Protein Implicated in Parkinson’s Disease

“…Human Fluorescent imaging was undertaken as previously described [12]. Briefly, transfected HEK293 were fixed and visualized for GFP fluorescence (Olympus IX70;…”