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Glatiramer Acetate (Copaxone®) is a Promising Gene Delivery Vector Nabil A Alhakamy, and Cory J. Berkland Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01282 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019
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Molecular Pharmaceutics
Glatiramer Acetate (Copaxone®) is a Promising Gene Delivery Vector
Nabil A. Alhakamy 1, 2 † and Cory J. Berkland, 2, 3* 1 Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah, KSA 2 Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA 66047 3 Department of Chemical & Petroleum Engineering, University of Kansas, Lawrence, KS, USA 66047
*Corresponding Author: Address: 2030 Becker Drive, Lawrence, KS 66047; Telephone: 785-864-1455; Fax: 785-864-1454; Email:
[email protected] †
Co-corresponding Author: King Abdulaziz University, Faculty of Pharmacy, Jeddah, KSA 21589; Telephone: (012)6400000/61955; Fax: (012)6951696; Email:
[email protected] 1
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Molecular Pharmaceutics
ABSTRACT Glatiramer acetate is the active substance of Teva’s Copaxone® drug, which contains random polypeptides used to treat multiple sclerosis (MS). Glatiramer acetate was originally developed to emulate human myelin basic protein, which contains four different residues [alanine (A), glutamic acid (E), tyrosine (T), and lysine (K)]. We found that Glatiramer acetate (GA) can complex, condense, and transfect plasmid DNA. Mixing the positively-charged GA and the negativelycharged genetic material at the correct proportions produced small, stable, and highly positivelycharged nanoparticles. This simple GA-pDNA formulation produced high levels of transfection efficiency with low toxicity in HeLa and A549 cells (lung and cervical cancer cells). Additionally, we studied and compared the nanoparticle properties, gene expression, and cytotoxicity of K100pDNA (high-molecular-weight polylysine) and K9-pDNA (low-molecular-weight polylysine) nanoparticles to GA-pDNA nanoparticles. We also studied the effect of calcium, which was previously reported to reduce the size and enhance gene expression resulting from similar polyelectrolyte complexes. Adding calcium did not reduce particle size, nor improve transfection efficiency of GA-pDNA nanoparticles as it did for polylysine-pDNA nanoparticles. GA-pDNA nanoparticles may be prepared by mixing a genetic payload with approved GA therapeutics (e.g., Copaxone®), thus offering intriguing possibilities for translational gene therapy studies.
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KEYWORDS: Glatiramer Acetate, Copaxone®, Gene Delivery, Polyelectrolyte, Transfection Efficiency, Polylysine, and Polyethylenimine.
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Molecular Pharmaceutics
INTRODUCTION Gene therapy has shown great promise for curing diseases (e.g., diabetes, hemophilia, infections, and tumors) through the insertion of therapeutic nucleic acids (e.g., siRNA, and pDNA) into target cells.1-3 Over the past 20 years, clinical applications of gene therapy have increased dramatically.1,
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Viral and nanoparticulate gene vectors, however, are complex pharmaceutical
products. Long and difficult development timelines are commonly required to identify formulations with sufficient stability and effective transfection of target tissues.1, 5 Perhaps more rapid progress could be made if simple formulations could be developed using already approved products.1 With this in mind, we explored glatiramer acetate (GA), the active substance from Teva’s Copaxone®, as a promising vector to complex and deliver plasmid DNA. The simplicity, efficiency, and safety of non-viral gene therapy vectors are one of the essential scientific challenges.6-9 Viral vectors (modified viruses: lentiviruses, adenoviruses, and retroviruses) have dominated the gene therapy clinical trials (~70%) for delivering genetic material.10, 11 Unfortunately, viral vectors are relatively complex and expensive to produce for clinical trials, have limited genetic material packaging capacity and pose a risk of immunogenicity and and carcinogenicity.2,
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Non-viral vectors are capable of overcoming some of these
limitations. Non-viral vectors could deliver larger genetic material payloads and may reduce immunogenicity. One of the greatest advantages is the relative ease with which non-viral vectors could be designed and prepared, compared to viral vectors.6, 11 GA, marketed by Teva Pharmaceuticals as Copaxone®, was officially authorized in 1996 by the US FDA for treating multiple sclerosis. GA is a combination of polypeptides consist of four different residues [alanine (A), lysine (K), glutamic acid (E), and tyrosine (T)] at a fixed molar ratio (0.427, 0.338, 0.141, and 0.095) (Table 1).12, 13 GA has an average molecular weight of 5000
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to 9000 Da and spans the range between 2500 and 20,000 Da.14-16 Since GA is approximately onethird lysine, we rationalized that GA may be useful as a polycation complexing agent to form nanoparticulate gene therapy vectors. The application of synthetic cationic peptides (e.g., polyarginine and polylysine) in pDNA or siRNA delivery have been widely studied. Synthetic high-molecular-weight polycations (e.g., ~20 kDa or higher) can complex and condense large pDNA or siRNA into steady and stable nanoparticles, but typically suffer from cytotoxicity.1,
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Conversely, low-molecular-weight
polycations (e.g.,
