Design and Screening of a Glial Cell-Specific, Cell Penetrating Peptide for Therapeutic Applications in Multiple Sclerosis

Abstract

Multiple Sclerosis (MS) is an autoimmune, neurodegenerative disease of the central nervous system (CNS) characterized by demyelination through glial cell loss. Current and proposed therapeutic strategies to arrest demyelination and/or promote further remyelination include: (i) modulation of the host immune system; and/or (ii) transplantation of myelinating/stem or progenitor cells to the circulation or sites of injury. However, significant drawbacks are inherent with both approaches. Cell penetrating peptides (CPP) are short amino acid sequences with an intrinsic ability to translocate across plasma membranes, and theoretically represent an attractive vector for delivery of therapeutic peptides or nanoparticles to glia to promote cell survival or remyelination. The CPPs described to date are commonly non-selective in the cell types they transduce, limiting their therapeutic application in vivo. Here, we describe a theoretical framework for design of a novel CPP sequence that selectively transduces human glial cells (excluding non-glial cell types), and conduct preliminary screens of purified, recombinant CPPs with immature and matured human oligodendrocytes and astrocytes, and two non-glial cell types. A candidate peptide, termed TD2.2, consistently transduced glial cells, was significantly more effective at transducing immature oligodendrocytes than matured progeny, and was virtually incapable of transducing two non-glial cell types: (i) human neural cells and (ii) human dermal fibroblasts. Time-lapse confocal microscopy confirms trafficking of TD2.2 (fused to EGFP) to mature oligodendrocytes 3–6 hours after protein application in vitro. We propose selectivity of TD2.2 for glial cells represents a new therapeutic strategy for the treatment of glial-related disease, such as MS.

Reappraisal of Human HOG and MO3.13 Cell Lines as a Model to Study Oligodendrocyte Functioning

“…Differentiation of MO3.13 with PMA yielded bipolar cells with a small cytoplasmic area, reminiscent of Schwann or astrocytic cells, while the N2.1 differentiation protocol produced a heterogeneous set of morphologies, including bipolar and multipolar cells. The four differentiation protocols applied here all increased MBP mRNA levels, which is in line with other reports [14,35,43], but we did not find MBP expression at the protein level, in agreement with other [32,43,60], but not all [40,47,50] studies on differentiated MO3.13 cells. Again, the issue of MBP mRNA-protein expression differences is not related to alternative splicing events, but remains otherwise unclear.…”

“…The HOG cells have been differentiated with N2 supplement (sodium selenite, thyroid hormone T3, insulin, transferrin) [7,8,14,15,16,17], N2 supplement complemented with putrescine, dibutyryl-cAMP (db-cAMP) and 3-isobutyl-1-methylxanthine (IBMX) [10,18,19,20,21,22,23] or N2 supplement complemented with D-biotin and progesterone [24,25,26,27]. Differentiation protocols for MO3.13 cells ranged from the use of phorbol 12-myristate 13-acetate (PMA) [8,12,13,14,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52] to serum-deprivation [9,53,54,55,56,57,58,59] and N2 supplement with combinations of progesterone, hydrocortisone, D-biotin and putrescine [7,14,25,34,60]. Furthermore, nearly all (~90%) differentiation studies on HOG and MO3.13 cells reported thus far did not use multiple markers and did not use myOL markers to phenotype the cells.…”

Designing and enhancing the antifungal activity of corneal specific cell penetrating peptide using gelatin hydrogel delivery system

“…Although nanoparticles, lipid vesicles, cell-penetrating peptides (CPPs), and CPP-conjugated therapies (CTTs) are now being used for topical drug delivery with high clinical efficacy, they lack cell specificity and bioavailability. 4 To address the issue of cell specificity, Heffernan et al 5 reported a method to generate glial-specific CPP for multiple sclerosis, from the surface proteins of lymphocytic choriomeningitis virus (LCMV) which infects glial cells. Bioavailability was enhanced by targeting conserved amino acid residues between antimicrobial peptide and CPP, 6 but none of them were able to generate tissuespecific peptides.…”

Stem Cell Therapy: A Promising Therapeutic Approach for Multiple Sclerosis

“…The potential of NSCs to differentiate into neuroglial cells and oligodendrocytes suggests their application as a beneficial method for the treatment of MS (79)(80)(81)(82)(83)(84). NSCs can also be derived from bone marrow, and these cells also exhibit the capacity for neuroglial differentiation (81,82).…”