Chapter 1. Delivery of Protein and Gene Therapeutics
1.1 Therapeutics Proteins and Protein Production Systems
As the “engines of life”, proteins play the most dynamic and diverse roles among all the macromolecules in the body, ranging from catalyzing biochemical reactions, controlling cell fates, forming cellular structures, providing tissue scaffolds, and transporting molecules within a cell or from one organ to another 1. On the one hand, owing to this functional specificity, disease may result when any one of these proteins contains mutations or other abnormalities, or is present in abnormally high or low concentrations, which pose an enormous challenge to modern medicine; Viewed from the perspective of therapeutics, on the other hand, the highly specific action of proteins makes them less likely to interfere with normal biological processes, and their high number represents a tremendous opportunity in terms of harnessing protein therapeutics to alleviate disease 2. In the early days, therapeutic proteins were isolated from natural biological sources and were not commonly used. For example, insulin was first purified from bovine pancreas in 1922 to treat diabetes 3, and growth hormone and the follicle-stimulating hormone were isolated from human pituitary glands 4,5. It was not until the early 1980s, with the advent of recombinant gene technologies, that the number of therapeutic applications of proteins started to explode remarkably. In 1982, the first recombinant pharmaceutical-human insulin came onto the market from Genentech6, and hitherto more than 130 proteins or peptides have been approved for clinical use by the FDA, with over 70% being recombinant, and many more are being developed world wide 2. Recombinant production offers distinct benefits: it provides drugs that could not have been made available by conventional methods; it manufactures proteins more efficiently and inexpensively, and in almost unlimited quantities; and it rules out certain possibilities of human pathogenic viruse contamination 7. Common heterologous proteins production systems include microbial fermentation, plant, insect and mammalian cell cultures. The Escherichia coli (E. coli) remains one of the most widely used hosts because of its easiest and quickest expression of proteins 8. However, the bacterium is not the system of choice to express very large proteins and proteins that require post-translational modifications. In such case, yeasts may be harnessed to produce eukaryotic heterologous proteins as they share similar subcellular post-translational protein modification machinery as eukaryotes 9. Nevertheless, for the production of therapeutic glycoproteins, yeasts are still less useful since their glycoproteins are associated with high mannose oligosaccharides that are readily recognized and sequestrated by macrophages bearing large numbers of mannose receptors on their surface 10. The mammalian cells have insofar become the dominant system for the production of recombinant versions of native proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification11.
1.2 Protein Engineering
Although recombinant proteins produced by mammalian cells are more biocompatible than those produced by other hosts, the full realization of their biomedical application is still largely restricted by poor catalytic activity, short shelf life and vulnerability to protease digestion. These drawbacks can be addressed with protein engineering to increase their clinical potential. Rational protein design and directed evolution are two general approaches of protein engineering 12. The rational protein design, which requires detailed knowledge of the structure and function of proteins, employs site-directed mutagenesis methods to make desired changes to proteins, yet sometimes the detailed structural knowledge is unavailable or incomplete. In sharp contrast, directed evolution relies on random mutagenesis followed by high-throughput screening to select variants having desired traits. The approach, however, entails immense screening efforts that even protein libraries with millions of members still sample a small fraction of possible sequence space for a protein 13. As a more recent approach, semi-rational methodologies combining the benefits of directed evolution and rational design are being used to significantly increase the efficiency of biocatalyst tailoring 12,13. As a result, a variety of improved enzyme properties have been attained, such as increased catalytic activity and stability 14-16, higher stereospecificity and stereoselectivity 14,17,18 altered pH profile 19,20, reduced immunogenicity 21,22 and enhanced inhibitor resistance 23.
Instead of just evolving proteins with altered properties, DNA shuffling and related methods are powerful tools in protein engineering to devise chimeric proteins with novel structures and properties. In particular, chimeric protein methodologies could contribute to unearthing potential novel roles of specific protein domains 24, preventing protein aggregation by altering net charge for a variety of applications 25,26, regenerating enzymatic cofactors simply and inexpensively 27,28, and facilitating protein purification 29. More importantly, the careful analysis of chimeric proteins promotes deeper understanding of protein structures, functions and key amino acid substitutions, which in return, could direct the rational protein redesign.
In Addition, impressive results of therapeutic proteins generated by other engineering approaches have also been reported, as evidenced by employing glycoengineering strategy to humanize yeast’s heterogeneous high mannose-type glycosylation to mammalian-type glycosylation to acquire pharmacokinetic stability and efficacy 30,31, and by utilizing the cell surface display and ribosome display approaches to engineer a variety of proteins for improved affinity, specificity, expression, stability and catalytic activity 32.
Despite the fact that as a powerful tool, protein engineering has overcome some of the limitations of biocatalysts, it is still flawed in a number of respects when it comes to the biomedical applications. First, as a trial and error methodology, it may still take considerable time to implement 13. Moreover, multiple functional domains of chimeric proteins may tangle together due to disadvantageous folding, leading to reduced activity and/or stability 33. Lastly, in protein engineering, protein intrinsic structure change was limited in order to maintain catalytic activity and stability, wherein alterations and improvements of major properties in terms of size, surface property and hydrophobicity are largely restricted.
To sum up, the past three decades have seen a drastic upsurge of engineered protein products on the market owing to the notable technical advances in recombinant DNA technology, high throughput genome sequencing, and protein engineering, providing a large potential for a vast scope of biomedical study and applications. In the field of precision medicine, these recombinant biologic agents nonetheless, are still at their primitive forms that require further decorations to accomplish the goal of a sustained and controlled release in vivo. Under this circumstances therefore, major properties such as size, charge, surface shape and surface hydrophobicity of these biologics need to be revisited and redressed.
1.3 Extracellular protein delivery
Nanotechnology, in its rough definition, refers to structures that span up to several hundred nanometers in size and that are developed by top-down or bottom-up engineering of individual components 34, Spurred by recent development in nanotechnology and drug delivery, Protein delivery has been actively pursued to treat a broad range of diseases including metabolic disorders 35,36, cancers 37 and tissue damage 38,39. Extracellular protein delivery, in particular, refers to delivering protein cargos to extracellular sites of action. Under these circumstances, lots of studies have been focusing on improving drug-loading capacity, targeting specificity, pharmaceutical efficacy and co-delivery issues. Many others have sought to modulate drug delivery vehicles to accomplish sustained drug release and stealth property.
It is worth noticing that the mononuclear phagocyte system (MPS) composed of dendritic cells, blood monocytes and tissue-resident macrophages is the primary phagocytosing system in the body strategically placed in many tissues. The cells are able to recognize and clear a multitude of invading foreign substances including particulates, cell debris, and microorganisms tagged with opsonin corona. As a result, evading MPS recognition and engulfment are crucial for exotic therapeutics to acquire prolonged in vivo half-life for extracellular protein delivery purposes40. In effect, there have been intensive studies showing that the biophysicochemical properties of a vehicle, such as size, charge, surface chemistry, and the nature and density of the ligands on their surface, have significant impacts on their clearance behavior and biodistribution 34. Typically, large-sized nanoparticles (>100 nm) tend to be more efficiently coated with opsonizing complement proteins leading to fast accumulation into the reticuloendothelial systems (RES) 41 42, whereas small nanoparticles with a final hydrodynamic diameter < 5.5 nm undergoes rapid urinary extraction and elimination from the body 43. Ideally, nanoparticles in the 10-100 nm size range can slow the renal clearance, splenic filtration and surface opsonization process 44. With respect to surface chemistry, charge and curvature, their combination could modulate the extent and type of opsonin binding in their coronas 45 46, which dictate subsequent cell uptake, gene expression, and toxicity 42 47. For example, Positively charged nanoparticles are cleared most quickly from the blood due to spontaneous labeling with negatively charged serum components 42. Neutral nanoparticles, instead, display highest blood half-life compared with charged ones 44. Hydrophobic particles have been shown to associate with blood serum opsonins more quickly than hydrophilic particles 40. Although many hurdles still exist for the development of nano-sized extracellular protein therapeutics that show sufficient blood circulation half-life, therapeutic efficiency, and no toxicity and immune response, their potential advantages should drive their successful development and translation into a new class of clinical therapies. To illustrate it, the following paragraphs exemplify several recent advancements of camouflaging or masking nanoparticles to temporarily bypass recognition by the MPS to order to increase blood or tissue resident time. 1.3.1 Poly (ethylene glycol) (PEG) –enzyme conjugates Conjugating polyethylene glycol (PEG) to proteins, known as PEGylation, is thus far the gold standard of stealth coating to prolong blood residence and reduce immunogenicity of therapeutic proteins.