Low back pain with resultant loss of function, decreased productivity, and high economic costs is burdensome for both the individual and the society. Evidence suggests that intervertebral disc pathology is a major contributor to spine-related pain and degeneration. When commonly used conservative therapies fail, traditional percutaneous or surgical options may be beneficial for pain relief but are suboptimal because of their inability to alter disc microenvironment catabolism, restore disc tissue, and/or preserve native spine biomechanics. Percutaneously injected Multipotent Mesenchymal Stem Cell (MSC) therapy has recently gained clinical interest for its potential to revolutionarily treat disc-generated (discogenic) pain and associated disc degeneration. Unlike previous therapies to date, MSCs may uniquely offer the ability to improve discogenic pain and provide more sustained improvement by reducing disc microenvironment catabolism and regenerating disc tissue. Consistent treatment success has the potential to create a paradigm shift with regards to the treatment of discogenic pain and disc degeneration.

A series of cationic amphiphlic copolymers (Z series) constructed from Tween 85 and low molecular weight (M w ) polyethyleneimine (LPEI) have been evaluated for the delivery of antisense 2 0 -O-methyl phosphorothioate RNA (2 0 -OMePS) in both cell culture and dystrophic mdx mice. All Z copolymers improved the 2 0 -OMePS-induced dystrophin expression both in vitro and in vivo compared with PEI 25k formulated or 2 0 -OMePS alone. The most effective polymers are in the order of Z9 4 Z3 4 Z7, Z1, Z2, Z6 4 others by formulation at the dose of 20 mg mL À1 in myoblast cell culture. Significantly enhanced exon-skipping of 2 0 -OMePS with Z polymers in mdx mice was obtained in the order of Z7 4 Z9, Z3 4 Z8, Z6 4 others. The highest efficiency of targeted exon-skipping with Z7 [T85-PEI 2k(1 : 1)] reached over 8 fold compared with 2 0 -OMePS alone in mdx mice. Further analyses of the structure and function indicate that the more hydrophobicity and lower PEI content of the polymer microstructure are, the greater are the delivery efficiency and exon-skipping. The unique hydrophobic interactions between the Z polymers and 2 0 -OMePS likely create more stable complexes in primarily hydrophilic environments both in vitro and in vivo. The overall results suggested that Tween 85 modified LPEIs provide a promising delivery approach for applications of 2 0 -OMePS oligonucleotides as therapeutic reagents.

Mutations in fukutin-related protein (FKRP) gene are characterized with lack of functionally glycosylated α-dystroglycan (F-α-DG). Surprisingly, a few muscle fibers express strong F-α-DG. Herein, we investigated the restoration of F-α-DG in the FKRP mutant muscles and showed that the restoration of glycosylation is associated with muscle regeneration and dependent on the expression of both like-glycosyltransferase (LARGE) and partially functional FKRP. F-α-DG in the regenerating fibers reaches up to normal levels and lasts for >4 weeks, but no up-regulation of the LARGE and FKRP is detected during the regeneration process. The FKRP protein with P448L mutation is sufficient for functional glycosylation of α-DG in regenerating fibers, but not in mature fibers. Thus, factors other than FKRP enable regenerating fibers to produce functional α-DG, compensating for the defect in FKRP function. Identification of factors other than LARGE and FKRP could generate new approaches for restoration of F-α-DG in mature muscle fibers with defects in FKRP functions.

A series of small-size polyethylenimine (PEI)-conjugated pluronic polycarbamates (PCMs) have been investigated for the ability to modulate the delivery of 2'-O-methyl phosphorothioate RNA (2'-OMePS) in vitro and in dystrophic mdx mice. The PCMs retain strong binding capacity to negatively charged oligomer as demonstrated by agarose gel retardation assay, with the formation of condensed polymer/oligomer complexes at a wide-range weight ratio from 1:1 to 20:1. The condensed polymer/oligomer complexes form 100-300 nm nanoparticles. Exon-skipping effect of 2'-OMePS was dramatically enhanced with the use of the most effective PCMs in comparison with 2'-OMePS alone in both cell culture and in vivo, respectively. More importantly, the effective PCMs, especially those composed of moderate size (2k-5kDa) and intermediate hydrophilic-lipophilic balance (7-23) of pluronics, enhanced exon-skipping of 2'-OMePS with low toxicity as compared with Lipofectamine-2000 in vitro or PEI 25k in vivo. The variability of individual PCM for delivery of antisense oligomer and plasmid DNA indicate the complexity of interaction between polymer and their cargos. Our data demonstrate the potential of PCMs to mediate delivery of modified antisense oligonucleotides to the muscle for treating muscular dystrophy or other appropriate myodegenerative diseases.