*Department of Inorganic and Analytical Chemistry Medical University of Warsaw, Faculty of Pharmacy, Warsaw Poland*

#### **1. Introduction**

138 Innovations in Biotechnology

Sanchez, M, Anitua, E, Azofra, J, et al. (2008). Intra-articular injection of an autologous

Sánchez, M; Anitua, E; Cugat, R; Azofra J, Guadilla J, Seijas R, Andia I. (2009). Nonunions

Sánchez M, Anitua E, Cole A, et al. (2009). Management of post-surgical Achilles tendon

Sanchez M, Anitua E, Azofra J, Prado R, Muruzabal F, Andia I. (2010). Ligamentization of

Sanchez, M, Anitua, E, Lopez-Vidriero, E, Andia, I. (2010). The Future: Optimizing the

Sanchez M, Guadilla J, Fiz N, Andia I. (2011). Ultrasound-guided platelet rich plasma

Sariguney, Y, Yavuzer, R, Elmas, C, et al. (2008). Effect of platelet-rich plasma on peripheral nerve regeneration. *Journal of Reconstructive Microsurgery* 24: 159-167. Schmikli SL, Backx FJG, Kemler HJ, van Mechelen W. (2009). National Survey on Sports

Sclafani AP. Platelet-rich fibrin matrix for improvement of deep nasolabial folds. (2010).

Semple JW, Freedman J. (2010). Platelets and innate immunity. *Cell Molecular Life Sciences*

Setta, HS, Elshahat, A, Elsherbiny, K, et al. (2011). Platelet-rich plasma versus platelet-poor

Soehnlein O, Zernecke A, Weber C. (2009). Neutrophils launch monocyte extravasation by

Villeneuve J, Block A, Le Bousse-Kerdiles MC, et al. Tissue inhibitors of matrix

Wee LL, Lee DYH, Soon MYH. (2009). Plasma Rich in Growth Factors to Treat Adductor Longus Tear. *Annals Academy of Medicine Singapore.* Aug 38(8):733-734. Whitman DH, Berry RL, Green DM. (1997). Platelet gel: an autologous alternative to fibrin

Wright-Carpenter T, Klein P, Schaferhoff P, Appell HJ, Mir LM, Wehling P. (2004).

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release granule proteins. *Thrombosis and Haemostasia* 02:198-205

cohort study. *Clinical Experimental Rheumatology* 26:910-3

*Trauma* 23 (1): 52-59

*Injury EXTRA* 40:11-15.

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67:499-511

Nov;55(11):1294-9

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doi:10.1093/rheumatology/ker303

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Treated With Autologous Preparation Rich in Growth Factors. *Journal of Orthopedic*

complications with a Preparation Rich in Growth Factors: A study of two-cases.

Tendon Grafts Treated With an Endogenous Preparation Rich in Growth Factors: Gross Morphology and Histology. *Arthroscopy-the Journal of Arthroscopic and Related*

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Biopolymers, synthetic polymers and their derivatives are commonly used in medicine and pharmacy. Significant progress attained in the polymer chemistry and technology has boosted the dynamic development of the medicinal engineering.

Recently, particular interest of scientists has been focused on biomedical polymers, especially those used for drug delivery systems, therapeutic systems and macromolecular prodrugs. The aforementioned applications have opened new exciting prospects for medicine, because specially designed polymers are capable of delivering medicinal substances to the target diseased tissues and cells together with dosing those drugs according to controlled specified pharmacodynamics. Particular attention has recently been paid to chemistry of biocompatiable and biodegradable polymers, because they have an advantage of being readily hydrolyzed into removable and non-toxic products, which can be subsequently eliminated by metabolic pathways. Furthermore, the biomedical polymers have to be synthesized now using friendly for the environment and safe for human health, effective natural initiators, co-initiators and/or catalysts.

Therefore, the main objective of this work is to discuss various polymers recommended for the pharmaceutical applications and then to describe natural compounds used as initiators, catalysts and co-initiators in the synthesis of biodegradable and bioresorbable polyesters and polycarbonates.

#### **2. Polymers in the pharmaceutical applications**

Macromolecules are applied in pharmacy as the pharmacological substances, blood substitutes, drug delivery and therapeutic systems, in the synthesis of macromolecular prodrugs and in the technology of prolonged release drug formulations.

#### **2.1 Polymers with the pharmacological effects and polymeric blood substitutes**

One of the most interesting polymers used in pharmacy, are those exerting a pharmacological effect. DIVEMA, copolymer of divinyl ether-maleic anhydride (Florjanczyk & Penczek, 1998; Papamatheakis et al., 1978) is an example of such compound with antitumoral and antiviral properties. Its action probably includes the stimulation of the glycoprotein production, which suppresses viral RNA translocation in cells and division of cancer cells.

Furthermore, the polymers are often applied as swelling, relaxation and sliding agents. Methylcellulose taken orally is not absorbed from the alimentary tract. However, it detains water on swelling and in consequence causes relaxation of the stercorous mass (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002).

A copolymer of ethylene and propylene glycols has found an application in the therapy of constipations (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). This non-ionic, surfaceactive polymer is unable to penetrate through the gut walls because of large average molecular weight. However, it causes relaxation and hydration of the stercorous mass by the reduction of the surface tension.

A linear polymer of uronic acids - alginic acid (mannuronic acid conjugated β-1,4 and Lguluronoic acid glycosidically conjugated α-1,4) is mainly obtained from the *Laminaria algae*. This polymer neutralizes hydrochloric acid (Janicki et al., 2002; Zejc & Gorczyca, 2002). Its action relies on detaining of water in stomach followed by reduction of irritations and pain.

A polyvinylpyrrolidone has found an application as anti-diarrhoeal drug (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). Its amphoteric properties normalize pH in stomach and intestines through acids or bases adsorption, which are usually raised as result of fermentation or putrefaction.

The synthetic hormones with the protein structure play an important role in the modern pharmacology (Zejc & Gorczyca, 2002). The Buserelin, Goserelin, Leuprorelin and Triptorelin are known as synthetic analogues of Gonadoliberin (the hormone of hypothalamus). These oligopeptides are obtained by exchanging of some amino acids in Gonadoliberin molecule and then are used to treat prostate and breast cancers or endometriosis. Another example is synthetic analogue of Somatoliberin used for treating children with some forms of GH deficiency. The synthetic analogue of Somatostatin - Octreotide is applied to treat the alimentary tract (Zejc & Gorczyca, 2002).

Corticotrophins are examples of synthetic hormones of the anterior pituitary, often applied in the therapy of rheumatoid diseases and severe asthma. Thus, Oxitocin, Vasopressin and Ornipressin are belong to the group of hormones of posterior lobe of the hypophysis (Zejc & Gorczyca, 2002). First of them causes uterine contractions, second can contract the smooth muscles of the blood vessels while Ornipressin is often added to the anaesthetics. Moreover causes the vessels contraction.

The peptide antibiotics are the relatively numerous group of the natural oligomers. They are composed of peptide-bounded amino acids to form cyclic, linear or cyclic-linear structures (Markiewicz & Kwiatkowski, 2001; Patrick, 2003; Zejc & Gorczyca, 2002). They may act the Gram-negative (Polymyxin) and Gram-positive (Gramicidin, Prostinamycin) bacteria as well as fungi and protozoa.

One of the most interesting polymers used in pharmacy, are those exerting a pharmacological effect. DIVEMA, copolymer of divinyl ether-maleic anhydride (Florjanczyk & Penczek, 1998; Papamatheakis et al., 1978) is an example of such compound with antitumoral and antiviral properties. Its action probably includes the stimulation of the glycoprotein production, which suppresses viral RNA translocation in cells and division of

Furthermore, the polymers are often applied as swelling, relaxation and sliding agents. Methylcellulose taken orally is not absorbed from the alimentary tract. However, it detains water on swelling and in consequence causes relaxation of the stercorous mass (Tonnesen &

A copolymer of ethylene and propylene glycols has found an application in the therapy of constipations (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). This non-ionic, surfaceactive polymer is unable to penetrate through the gut walls because of large average molecular weight. However, it causes relaxation and hydration of the stercorous mass by

A linear polymer of uronic acids - alginic acid (mannuronic acid conjugated β-1,4 and Lguluronoic acid glycosidically conjugated α-1,4) is mainly obtained from the *Laminaria algae*. This polymer neutralizes hydrochloric acid (Janicki et al., 2002; Zejc & Gorczyca, 2002). Its action relies on detaining of water in stomach followed by reduction of irritations and pain. A polyvinylpyrrolidone has found an application as anti-diarrhoeal drug (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). Its amphoteric properties normalize pH in stomach and intestines through acids or bases adsorption, which are usually raised as result of

The synthetic hormones with the protein structure play an important role in the modern pharmacology (Zejc & Gorczyca, 2002). The Buserelin, Goserelin, Leuprorelin and Triptorelin are known as synthetic analogues of Gonadoliberin (the hormone of hypothalamus). These oligopeptides are obtained by exchanging of some amino acids in Gonadoliberin molecule and then are used to treat prostate and breast cancers or endometriosis. Another example is synthetic analogue of Somatoliberin used for treating children with some forms of GH deficiency. The synthetic analogue of Somatostatin -

Corticotrophins are examples of synthetic hormones of the anterior pituitary, often applied in the therapy of rheumatoid diseases and severe asthma. Thus, Oxitocin, Vasopressin and Ornipressin are belong to the group of hormones of posterior lobe of the hypophysis (Zejc & Gorczyca, 2002). First of them causes uterine contractions, second can contract the smooth muscles of the blood vessels while Ornipressin is often added to the anaesthetics. Moreover

The peptide antibiotics are the relatively numerous group of the natural oligomers. They are composed of peptide-bounded amino acids to form cyclic, linear or cyclic-linear structures (Markiewicz & Kwiatkowski, 2001; Patrick, 2003; Zejc & Gorczyca, 2002). They may act the Gram-negative (Polymyxin) and Gram-positive (Gramicidin, Prostinamycin) bacteria as well

Octreotide is applied to treat the alimentary tract (Zejc & Gorczyca, 2002).

**2.1 Polymers with the pharmacological effects and polymeric blood substitutes**

cancer cells.

Karlsen, 2002; Zejc & Gorczyca, 2002).

the reduction of the surface tension.

fermentation or putrefaction.

causes the vessels contraction.

as fungi and protozoa.

A cyclosporine A – branched and cyclic oligopeptide composed of 11 amino acids is an important macromolecular immunosuppressive drug (Markiewicz & Kwiatkowski, 2001; Zejca & Gorczyca, 2002). Cyclosporine A selectively inhibits lymphocytes T function, thus is widely used as an immune barrier tolerance agent in the transplantology.

Macromolecular inhibitors that absorb the cholesterol from the intestines are also known; form them insoluble in water polymers, which produce complexes with the bile acids. To this polymer group belongs: copolymer of divinylbenzene and styrene substituted with quaternary trimethylammonium group and copolymer of diethyltriamine and epichlorohydrin (Zejc & Gorczyca, 2002).

A heparin, obtained from the animal tissues (mainly livers and lungs) (Zejc & Gorczyca, 2002) is next example of the natural polysaccharide used as the therapeutic agent. The heparin effects on the all blood clotting phases. Usually is used to treat arterial embolism and thrombosis, heart failure and before surgical operations.

A very important group of the biomedical polymers is macromolecular blood substitutes. They are accountable for the regular osmotic pressure and viscosity, closed to the osmotic pressure and blood viscosity; usually used in the anaphylactic shock, heart failure, intoxication, burns, toxic diarrhoea, embolic-thrombotic complications as well as microcirculation impairment.

A polyvinylpyrrolidone was the first synthetic polymer used as the blood substitutes. Its solutions were mainly used to treat the shock after the burns and, in the case when the blood transfusion was not indicated (Janicki et al., 2002; Florjanczyk & Penczek, 1998; Zejc & Gorczyca, 2002). Likewise, the solutions of polyvinyl alcohol have found the applications as the blood substitutes. However, they were withdrawn from the list of the blood substitutes as result of their undesirable side effects.

The blood susbstitutes with the therapeutic action has also been elaborated as result of incorporation of some therapeutic agents (e.g. penicillin, pelentanic acid, p-aminosalicylic chloride) into polyvinyl alcohol (Janicki et al., 2002; Zejc & Gorczyca, 2002).

Currently, the solutions of polysaccharides (e.g. dextran), modified starch derivatives and modified gelatin products (polygeline, oksopolygelin, liquid gelatin) are commonly used as the blood substititues (Janicki et al., 2002; Zejc & Gorczyca, 2002). The dextran with the average molecular weight ranged from 40 000 to 70 000 Da is used as 6 or 10% solution. This polysaccharide is produced by fermentation of the sucrose solutions in the presence of the *Leuconostoc mesenteroides* bacteria. Obtained glucose is polymerized to dextran in the presence of enzymes.

A hydroksyethyl starch is obtained by hydrolysis of high-amylopectine starch in acidic environment (Zejc & Gorczyca, 2002). The reaction products are neutralized followed by the reaction with ethylene oxide. The starch substituted with hydroxyethyl group is then produced in this reaction.

A polygeline is obtained from the reaction of diisocyanate with the gelatin. As result, linked urea groups are produced, whereas liquid gelatin is produced in the reaction with succinic anhydride (Janicki et al., 2002; Zejc & Gorczyca, 2002).

#### **2.2 Macromolecular prodrugs**

A prodrug is a modified therapeutic agent, which is metabolized into active precursor in human body (Janicki et al., 2002). Over the recent years, the conception of macromolecular prodrug has appeared as macromolecule that has therapeutic agents in the structure; the released drug becomes pharmacologically active during hydrolytic biodegradation of the polymer (Ouchi & Ohya, 1995). In general, the therapeutic agent could be incorporate into polymer chain, might be end-capped or may form a pendant group of the macromolecular chain (Figure 1).

Fig. 1. Structure of the macromolecular prodrugs

A prodrug is a modified therapeutic agent, which is metabolized into active precursor in human body (Janicki et al., 2002). Over the recent years, the conception of macromolecular prodrug has appeared as macromolecule that has therapeutic agents in the structure; the released drug becomes pharmacologically active during hydrolytic biodegradation of the polymer (Ouchi & Ohya, 1995). In general, the therapeutic agent could be incorporate into polymer chain, might be end-capped or may form a pendant group of the macromolecular

**2.2 Macromolecular prodrugs**

Fig. 1. Structure of the macromolecular prodrugs

chain (Figure 1).

Macromolecular prodrugs are mainly used in the cancer therapy. For example, 5 fluorouracil can be applied locally or orally in the therapy of the alimentary tract, urinary bladder and prostate gland cancers. The conjugations of this therapeutic agent as a pendant group to polyethylene glycol (Ouchi et al., 1986, 1992) or to vinyl polymer chain as substituent form examples of its macromolecular prodrugs (Ouchi et al., 1988).

The pharmacokinetics of the macromolecular prodrugs is mainly determined by the structure of the polymer (the rate of hydrolysis under the given conditions and the susceptibility to degradation in the presence of enzymes), its average molecular weight (the ability to the accumulation in blood, lymph, spleen, liver and other organs) or crystallinity (the rate of biodegradation).

The polymers must meet specific criteria to be applied in the synthesis of the macromolecular prodrugs. Namely, macromolecules and their metabolic decomposition products cannot be cumulate in the human body, to be toxic and the most important; the drug should be released from the macromolecule as result of the metabolic processes. The list of the macromolecular prodrugs developed so far is broadly presented in monograph (Ouchi & Ohya, 1995).

#### **2.3 Polymers in the technology of prolonged release drug formulations**

Macromolecules have also found the application in the technology of prolonged release drug formulations. They are mainly intended to ensure the constant concentration of the therapeutic agent in the certain time (e.g. 8-24 hours), in the patient body. The group of these drugs, therefore, can eliminate the drug multiple dosing during a day and reduce total daily dose of it. The prolonged drug forms are usually applied in the therapy of cardiac and alimentary tract diseases, coronary vessels, diabetics, and psychiatric disorders.

The absorption of the therapeutic agent using prolonged release drug forms can be reduced by coating, incorporation, complexation or bonding on the ionites (Janicki et al., 2002). Polymers applied in this technology, could be generally divided into biodegradable and non-biodegradable. Biodegradable macromolecules are definitely more preferred from the toxicological point of view. In the technology of prolonged release drug formulation, natural polymers and their modified derivative (e.g.: starch, cellulose) as well as synthetic polymers are used e.g.: polyethylene, polypropylene, polyvinyl chloride, polyvinyl alcohol, polyvinyl acetate, polyacrylic acid, polycarbophile, polyacrylamides, polyacrylates, polyethylene glycol, poly(amino amide)s, polyurethanes, siloxanes, homoor copolymers of lactide and glycolide, poly(ε-caprolactone), polyorthoesters (Cardamone et al., 1997; Ertan et al., 1997; Huang et al., 1994; Lan et al., 1996; Matthews et al., 1996; Merkli et al., 1998; Ouchi & Ohya, 1995; Schierholz 1997; Sintzel et al., 1996; Uhrich et al., 1999; Ulbrich et al., 1996).

The crystals, pellets and granules of the drug might be coated with several polymer layers, according to the expected release rate. The therapeutic agent is gradually released as result of the polymer erosion or diffusion or is rinsing out from the polymer coating (Figure 2) (Uhrich et al., 1999).

Methylcellulose, polyvinylpyrrolidone and polyvinyl alcohol are predominantly applied as the coating substances. The analogous effect can be obtained by coating of the therapeutic agent with polymeric layers, soluble in different parts of the alimentary tract or under enzymes.

The drug release based on the diffusion takes place when polymers insoluble in the alimentary tract (e.g.: ethyl cellulose, nitrocellulose, cellulose acetate, acrylic and methacrylic ester copolymers) are applied as the coating agents. The coating tablets containing porophors (acrylic and methacrylic ester copolymers, starch, cellulose acetate phthalate or microcrystalline cellulose) are also used. The solubility of these tablets is increased as the effect of porophors dissolution and swelling.

Fig. 2. The mechanism of the controlled release of the therapeutic agent

The incorporation method is relying on the suspension of the therapeutic agent on the prolonged released carrier. Most often as the carriers are used: hydrophobic polymers (e.g.: methylcellulose, acrylic acid polymers) as well as lipopholic polymers and some carriers insoluble in the alimentary tract (e.g.: polyvinyl chloride, polyethylene, cellulose acetate, ethyl cellulose, polystyrene, polyamide, silicone resin and acrylic and metacrylic acids ester copolymers). For instance, when the hydrophilic carrier is used, the tablet is consecutively swelled after passing the alimentary tract followed by creation of high viscous hydogels, which prolonged the drug release. The drug release suspended on the lipophilic carrier is dependant on pH and the presence of enzymes. Matrix tablets contained water-insoluble carriers, however, are stable in the alimentary tract environment. Therefore, the drug is gradually release via the capillaries.

The complexation method involves the creation of poor soluble, therapeutic agent-polymer complexes. The drug is released due to the gradual decomposition of this complex. This technique is also used to produce skin and mucosa antiseptics (iodophors). The iodophors are the complexes of iodine with water-soluble polymers, which perform a role of carrier. They are high active against bacteria, viruses, fungi and protozoa.

The bonding of the drug on the ionites method is usually applied for acidic or basic drugs. It relies on release of the drug based on ion exchange in the alimentary tract.

#### **2.4 Polymers in the therapeutic systems technology**

144 Innovations in Biotechnology

therapeutic agent with polymeric layers, soluble in different parts of the alimentary tract

The drug release based on the diffusion takes place when polymers insoluble in the alimentary tract (e.g.: ethyl cellulose, nitrocellulose, cellulose acetate, acrylic and methacrylic ester copolymers) are applied as the coating agents. The coating tablets containing porophors (acrylic and methacrylic ester copolymers, starch, cellulose acetate phthalate or microcrystalline cellulose) are also used. The solubility of these tablets is increased as the

or under enzymes.

effect of porophors dissolution and swelling.

gradually release via the capillaries.

Fig. 2. The mechanism of the controlled release of the therapeutic agent

The incorporation method is relying on the suspension of the therapeutic agent on the prolonged released carrier. Most often as the carriers are used: hydrophobic polymers (e.g.: methylcellulose, acrylic acid polymers) as well as lipopholic polymers and some carriers insoluble in the alimentary tract (e.g.: polyvinyl chloride, polyethylene, cellulose acetate, ethyl cellulose, polystyrene, polyamide, silicone resin and acrylic and metacrylic acids ester copolymers). For instance, when the hydrophilic carrier is used, the tablet is consecutively swelled after passing the alimentary tract followed by creation of high viscous hydogels, which prolonged the drug release. The drug release suspended on the lipophilic carrier is dependant on pH and the presence of enzymes. Matrix tablets contained water-insoluble carriers, however, are stable in the alimentary tract environment. Therefore, the drug is

The complexation method involves the creation of poor soluble, therapeutic agent-polymer complexes. The drug is released due to the gradual decomposition of this complex. This technique is also used to produce skin and mucosa antiseptics (iodophors). The iodophors The polymers used in the therapeutic systems are the drug forms that are dosing or releasing drug in the exact time with the controlled rate (Janicki et al., 2002; Müller & Hildebrand, 1998). They are designed to ensure constant concentration of the therapeutic agent in the body (Figure 3).

Fig. 3. Drug release profile from the conventional tablets and polymeric therapeutic systems

Therapeutic systems are commonly used in medicine due to their high efficiency in comparison to the conventional drug forms and prolonged release tablets. Considering the way of administration and the location of the drug absorption there are: oral, transdermal, ocular, intra-uterine, implantation and infusion therapeutic systems (Table 1). According to the construction of the element dosing the drug, there are: membrane, matrix and microvessels transdermal therapeutic systems (Knoch & Merkle, 1987; Müller & Hildebrand, 1998), usually used to treat stenocardia, inflammations, motion sickness, chronic hypertensive disease, in the hormonal and anti-nicotinic therapies (De Mey et al., 1989; Fagerström et al., 1993; Hadgraft, 1996; Ho & Chien, 1993; Liedtke et al., 1989; Lin et al., 1993; Man et al., 1993; Monkhouse & Hug, 1988; Sanders, 1996). The novelty comprises the ultrasonic transdermal therapeutic systems and the microelectronics transdermal therapeutic systems, where the drug is released from the polymer carrier under the frequency electric field influence (Prausnitz et al., 1994; Santus & Baker, 1993; Simonin, 1995).

In the ocular therapeutic systems, the drug is released to the lachrymal fluid through the membrane. The intra-uterine therapeutic systems are mostly used in the contraception, whereas implantation therapeutic systems are usually applied under the skin. In their case, the drug release is carried out through the slow diffusion from the polymeric systems to the tissue.

Table 1. The therapeutic systems examples

#### **3. Natural and bioactive initiators and catalysts in the synthesis of biodegradable and bioresorbable polyesters and polycarbonates**

Biodegradable and bioresorbable polymers such as polyglycolide (PG), polylactide (PLA), poly(ε-caprolactone) (PCL), poly(trimethylene carbonate) (PTMC) and copolymers of glycolide (GL), L-lactides (LA), *rac*-lactide (*rac*-LA), ε-caprolactone (CL), trimethylene carbonate (TMC) or others cyclic esters and carbonates are very often used as polymeric prodrugs, drug delivery or therapeutic systems. Aliphatic polyesters and polycarbonates are degraded *in vivo* by hydrolytic deesterification into glycolic, lactic or other acid monomers. The latter species become involved in the carboxylic acid cycle and are subsequently excreted as carbon dioxide and water. Furthermore, biodegradable and bioresorbable drug forms exhibit unique pharmacokinetics, body distribution and pharmacological efficacy.

In the ocular therapeutic systems, the drug is released to the lachrymal fluid through the membrane. The intra-uterine therapeutic systems are mostly used in the contraception, whereas implantation therapeutic systems are usually applied under the skin. In their case, the drug release is carried out through the slow diffusion from the polymeric

**Therapeutic system Polymer Drug**

copolymers of acetate vinyl and ethyl, poliacrylate, silicone, polyurethanes, polyolefines, polyethylene glycol

polyvinyl alkohol, polyacetale vinyl, polyamides, polyethylene glycol, poliacrylate, silicone, homo- or copolymers of lactide, glicolide and ε-

system copolymers of acetate vinyl and ethyl Pilocarpina

system silicone Progesterone

**3. Natural and bioactive initiators and catalysts in the synthesis of biodegradable and bioresorbable polyesters and polycarbonates**

copolymers of lactide and glicolide,

Biodegradable and bioresorbable polymers such as polyglycolide (PG), polylactide (PLA), poly(ε-caprolactone) (PCL), poly(trimethylene carbonate) (PTMC) and copolymers of glycolide (GL), L-lactides (LA), *rac*-lactide (*rac*-LA), ε-caprolactone (CL), trimethylene carbonate (TMC) or others cyclic esters and carbonates are very often used as polymeric prodrugs, drug delivery or therapeutic systems. Aliphatic polyesters and polycarbonates are degraded *in vivo* by hydrolytic deesterification into glycolic, lactic or other acid monomers. The latter species become involved in the carboxylic acid cycle and are subsequently excreted as carbon dioxide and water. Furthermore, biodegradable and bioresorbable drug forms exhibit unique pharmacokinetics, body distribution and

silicone Estradiol, Goserelin

caprolactone

Acetate Noretisterone, Buprenorphine, Clonidyne,

Estradiol, Fentanyl, Flurbiprofen, Hyoscine, Isosorbide dinitrate,

Nicotyne, Nitroglycerin, Testosterone

Acetazolamide, Glipizide, Metoprolol,

Nifedipine, Okseprenolol KCl, Li2SO4, FeSO4

systems to the tissue.

Transdermal therapeutic system

Oral therapeutic

Ocular therapeutic

Uterus therapeutic

pharmacological efficacy.

Table 1. The therapeutic systems examples

Implantation therapeutic system

system

There are two methods of the synthesis of aliphatic polyesters or polycarbonates, namely polycondensation of diols, dicarboxylic acids or hydroxycarboxylic acids and ring-opening polymerization (ROP) of cyclic monomers (Platel, 2009; Labet & Thielemans, 2009). The polycondensation is hampered by typical limitations of step polymerization. The polymers obtained in this process are characterized by a high polydispersity. ROP gives polymeric products with the higher molecular weight and lower polydispersity. Therefore, is more preferred route to obtain aliphatic polyesters or polycarbonates (Platel, 2009).

The ROP of cyclic esters, carbonates or ether-esters initiated or catalyzed by the metal complexes or organic compounds yields high molecular weight polymers with the excellent conversion. The metal compounds are used commercially due to their selectivity, rate and lack of side reactions. On the other hand, for some biomedical or pharmaceutical applications, metal residues (Zn, Al, Sn) are undesirable (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Varma et al., 2005).

#### **3.1 Natural catalysts of ring-opening polymerization of cyclic esters and carbonates**

The application of enzymes as catalysts of ROP seems to be a perspective direction in the polymer research. Macromolecules with well-defined structures can be formed by enzymecatalyzed processes. On the other hand, the use of enzymes has some disadvantages, such as high cost, large quantity of enzymes required for ROP and relatively low molecular weight of the obtained polymers. However, the metal-free method of polymerization and suitable molecular weights of the resulted polymers are desirable conditions for the pharmaceutical applications, especially for the design of new drug delivery systems.

Fig. 4. e-ROP of cyclic esters, ether-esters or carbonates

The primary research on the enzyme ring-opening polymerization (e-ROP) has been carried out for CL. Currently major works concern ROP of six- and seven-membered cyclic esters, cyclic ether-estres or carbonates.

Lipases could also catalyze ROP of cyclic monomers, with different ring sizes as well as monomers containing substituents in the ring: α-methyl-β-propiolactone (1), β–butyrolactone (2), γ-caprolactone (3), α-methyl-valerolactone (4), 1,4-dioxan-2-one (5), δ-caprolactone (6), γethyl-ε-caprolactone (7), ε-heptanolactone (8), δ-decalactone (9), δ-dodecalactone (10), αmethyl-12-dodecanolide (11), α-methyl-15- pentadecanolide (12), L-lactide (13), D-lactide (14), D,L-lactide (15), 1,4-dioxepan-2-one (16), 1,5-dioxepan-2-one (17), 2-methylene-4-oxa-12 dodecanolide (18), 1,3-dioxan-2-one (19), 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (20), 5-benzyloxy-1,3-dioxan-2-one (21), 1-methyl-1,3-dioxan-2-one (22), cyclobis (hexamethylene carbonate) (23), 2,2'-dimethyl-1,3-dioxan-2-one (24) (Figure 5) (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009; Varma et al., 2005).

Lipases can accommodate a wide variety of synthetic substrates and still be able to show stereo- and regio-selectivity. They have evolved unusually stable structures that may survive effect of the organic solvents. The lipase-catalyzed hydrolysis in water can be easily reversed in non-aqueous media or bulk into ester synthesis or transesterification (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009; Varma et al., 2005).

The e-ROP can be carried out in bulk, in organic media and at various interfaces. Enzymecatalyzed reactions proceed under different reaction conditions (i.e. temperature, pressure, time). As an example, e-ROP of cyclic monomers was performed using lipase as catalyst for 2-720 h. *M*n of the resulting polymers was ranged from 1000 to 90 000 Da, when *M*w was in the range from 6 000 to 170 000 Da. The yield of the obtained polymers varied from 10 to 100%. The preferred lipase system generally used is a physically immobilized form of *Candida Antarctica*, commercially available as Novozyme-435 (Barrera-Rivera et al., 2009; Córdova et al., 1999; Divakar, 2004; Dong, 1998, 1999; Gorke et al., 2007; Henderson et al., 1996; Kobayashi, 2001a, 2001b, 2009; MacDonald et al., 1995; Marcilla et al., 2006; Matsumoto et al., 1999; Mei et al., 2003; Namekawa et al., 1999; Sivalingam & Madras, 2004; Van Der Mee et al., 2006).

The primary research on the enzyme ring-opening polymerization (e-ROP) has been carried out for CL. Currently major works concern ROP of six- and seven-membered cyclic esters,

Lipases could also catalyze ROP of cyclic monomers, with different ring sizes as well as monomers containing substituents in the ring: α-methyl-β-propiolactone (1), β–butyrolactone (2), γ-caprolactone (3), α-methyl-valerolactone (4), 1,4-dioxan-2-one (5), δ-caprolactone (6), γethyl-ε-caprolactone (7), ε-heptanolactone (8), δ-decalactone (9), δ-dodecalactone (10), αmethyl-12-dodecanolide (11), α-methyl-15- pentadecanolide (12), L-lactide (13), D-lactide (14), D,L-lactide (15), 1,4-dioxepan-2-one (16), 1,5-dioxepan-2-one (17), 2-methylene-4-oxa-12 dodecanolide (18), 1,3-dioxan-2-one (19), 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (20), 5-benzyloxy-1,3-dioxan-2-one (21), 1-methyl-1,3-dioxan-2-one (22), cyclobis (hexamethylene carbonate) (23), 2,2'-dimethyl-1,3-dioxan-2-one (24) (Figure 5) (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009; Varma et al., 2005).

 Lipases can accommodate a wide variety of synthetic substrates and still be able to show stereo- and regio-selectivity. They have evolved unusually stable structures that may survive effect of the organic solvents. The lipase-catalyzed hydrolysis in water can be easily reversed in non-aqueous media or bulk into ester synthesis or transesterification (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009;

The e-ROP can be carried out in bulk, in organic media and at various interfaces. Enzymecatalyzed reactions proceed under different reaction conditions (i.e. temperature, pressure, time). As an example, e-ROP of cyclic monomers was performed using lipase as catalyst for 2-720 h. *M*n of the resulting polymers was ranged from 1000 to 90 000 Da, when *M*w was in the range from 6 000 to 170 000 Da. The yield of the obtained polymers varied from 10 to 100%. The preferred lipase system generally used is a physically immobilized form of *Candida Antarctica*, commercially available as Novozyme-435 (Barrera-Rivera et al., 2009; Córdova et al., 1999; Divakar, 2004; Dong, 1998, 1999; Gorke et al., 2007; Henderson et al., 1996; Kobayashi, 2001a, 2001b, 2009; MacDonald et al., 1995; Marcilla et al., 2006; Matsumoto et al., 1999; Mei et al., 2003; Namekawa et al., 1999;

Sivalingam & Madras, 2004; Van Der Mee et al., 2006).

cyclic ether-estres or carbonates.

Varma et al., 2005).

Fig. 5. Representative monomers for e-ROP

The mechanism of e-ROP of cyclic esters using lipases as catalyst has been proposed by several authors. Monomer activated e-ROP (Scheme 1) involves the activation of the monomer molecules by an enzyme followed by the attack of the activated monomer onto the polymer chain end (Albertsson & Srivastava, 2008; MacDonald et al., 1995; Namekawa et al., 1999).

The ROP of cyclic carbonates catalyzed by enzyme or enzyme derivatives, in which polyesters, poly(ether-ester)s and polycarbonates terminated by hydroxyl groups are obtained, seems very attractive from the pharmaceutical or medical point of view.

Bisht and coworkers proposed a mechanism for chain initiation and propagation for lipasecatalyzed trimethylene carbonate polymerization, based on the symmetrical structure of these products and the end-group structure of high molecular weight chains (Scheme 2) (Bisht et al., 1996).

Scheme 2. The mechanism of e-ROP of cyclic carbonates

Bisht and coworkers proposed a mechanism for chain initiation and propagation for lipasecatalyzed trimethylene carbonate polymerization, based on the symmetrical structure of these products and the end-group structure of high molecular weight chains (Scheme 2)

Scheme 1. The mechanism of e-ROP of cyclic esters

Scheme 2. The mechanism of e-ROP of cyclic carbonates

(Bisht et al., 1996).

#### **3.2 Natural initiators and organocatalysts of ring-opening polymerization of cyclic esters and carbonates**

Recently, many modification approaches on biodegradable and bioresorbable polymers were carried out to meet the requirements of specific medical and pharmaceutical applications. Between them, incorporation of bioactive or biocompatible compounds such as lipids, amino acids into polymer chain or using of natural products as organocatalysts has received considerable attention.

The guanidine is a natural base, existing in human body and some vegetables. Some guanidine derivatives are the components of the therapeutic agents (Kinnel et al., 1998; Ramarao et al., 1993). Application of guanidine derivatives as organocatalysts for the synthesis of biodegradable polymers is an attractive way in the materials science technology. Li and coworkers reported the use of hexabutyl guanidinum acetatae in the living ROP of lactides (LAs) (Li et al., 2004). The polymerization was performed in bulk, producing polylactides (PLAs) with moderate molecular weight and narrow polydispersity. Strong guanidine bases: TBD (1,5,7-Triazabicyclo[4.4.0]dec-5-ene), MTBD (7-Methyl-1,5,7 triazabicyclo[4.4.0]dec-5-ene) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) were applied as effective organocatalysts for ROP of LA, δ-valerolactone (VL) and CL by Lohmeijer`s and coworkers (Lohmeijer et al., 2006). They found that TBD was polymerized LA, VL and CL in the controlled manner while MTBD and DBU polymerized only LA. For VL and CL the addition of thiourea co-catalyst was required. Wang and coworkers were used creatinine as catalysts of ROP of LA, examining the influence of temperature, time and creatinine dosage on the polymerization and properties of the produced biodegradable polymer (Wang et al., 2003). Based on the obtained results they proposed that creatinine is initiated ROP of LA according to the coordination-insertion mechanism. The biogenetic guanidine carboxylates: creatinine acetate (CRA) and creatinine glycolate (CRG) were synthesized and then effectively utilized as single-component initiators of ROP of LAs (Li et al., 2009). The mechanism of ROP was proposed based of the experimental investigation. In our laboratory, other guanidine derivatives: arginine and citrulline were successfully applied as initiators of ROP of LA and CL (Oledzka et al., 2011). The incorporation of α-amino acid molecules into the polymer chain was confirmed using 1H, 13C NMR and FT-IR spectroscopy and MALDI TOF MS spectrometry.

Various carboxylic acids (lactic, tartaric, hexanoic, propionic and citric acids) and natural amino acids (glycine, proline and serine) were engaged as catalysts in living ROP of CL and VL (Casas et al., 2004). The reactions were performed without solvent with the efficient way of recovering of the catalysts. Moreover, the authors found that the order of catalytic efficiency of the organic acid catalysts in ROP was as follows: tartaric acid (pKa=2.98)>citric acid (pKa=3.08)>lactic acid (pKa=3.14)>proline (pKa=1.95).

The fatty acids are found naturally in the human body. They are considered biologically safe and are generally considered suitable candidates for the preparation of biodegradable polymers (Teomim & Domb, 2001).

ROP of CL by organic acids catalyst and oleic acid derivatives initiator systems was investigated by Oledzka and coworker (Oledzka & Narine, 2001). They have found that the polymerizations were efficiently catalyzed by succinic and fumaric acid. The incorporation of fatty acid molecules resulted in less crystallinity and lower melting points of the obtained polymer samples. Furthermore, the degradation rate of the received polymers was slower when compared to CL homopolymer, but increase in porosity of the polymers was observed over time.

Natural amino acids are essential components in human nutrition. The studies performed by Liu and coworkers showed that they are also effective as initiators of ROP of CL (Liu & Liu, 2004). Authors found hat the number-average molecular weights of the obtained polymers did not exceed 5700 Da and that used amino acids were incorporated into macromolecule chain (Figure 6). In our laboratory, more detailed studies, involving an extended range of amino-acid initiators applied to ROP of CL and *rac*-LA were previously performed (Sobczak et al., 2008). The polymerization of CL and *rac*-LA was carried out in bulk, at 120–160 ºC. L-alanine, Lcysteine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-aspartic acid, Lglutamic acid, L-histidine, L-lysine were used as initiators of ROP. Aliphatic polyesters have synthesized with high yield (even ca. 100 % in some cases).

Fig. 6. ROP of cyclic esters using natural amino acids as initiators

ROP of aliphatic cyclic carbonates using natural amino acids was also investigated by Liu and coworkers (Liu et al., 2008). The biodegradable polymers with incorporated amino acids molecules were effectively obtained. The presence of natural amino acids in the polymer chain was proved by nuclear magnetic resonance spectroscopy.

Polyamidoamine (PAMAM) dendrimers are highly hyperbranched synthetic polymers with well-defined structure that allows accurate control of shape, size and functionality of terminal groups (Tomalia et al., 1985). The PAMAM dendrimers have useful applications in pharmaceutical technology e.g. as carriers in drug delivery systems, which can cross cell walls by both paracellular and transcellular pathways (Jevprasesphant et al., 2003). In our laboratory, novel star-shaped biodegradable polyesters were synthesized by ROP of CL using PAMAM dendrimer initiator without any metal catalysts (Oledzka et al., 2011). The nonlinear structure as well as physicochemical properties of the obtained biodegradable polymers were confirmed by nuclear magnetic resonance, gel permeation chromatography, thermal gravimetric analysis and differential scanning calorymetry.

#### **3.3 Natural co-initiators of ring-opening polymerization of cyclic esters, ether-esters and carbonates**

Metal catalyzed ROP of cyclic esters, ether-esters or carbonates has become the object of intensive studies with respect to their practical applications in the synthesis of biomaterials.

polymer samples. Furthermore, the degradation rate of the received polymers was slower when compared to CL homopolymer, but increase in porosity of the polymers was

Natural amino acids are essential components in human nutrition. The studies performed by Liu and coworkers showed that they are also effective as initiators of ROP of CL (Liu & Liu, 2004). Authors found hat the number-average molecular weights of the obtained polymers did not exceed 5700 Da and that used amino acids were incorporated into macromolecule chain (Figure 6). In our laboratory, more detailed studies, involving an extended range of amino-acid initiators applied to ROP of CL and *rac*-LA were previously performed (Sobczak et al., 2008). The polymerization of CL and *rac*-LA was carried out in bulk, at 120–160 ºC. L-alanine, Lcysteine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-aspartic acid, Lglutamic acid, L-histidine, L-lysine were used as initiators of ROP. Aliphatic polyesters have

ROP of aliphatic cyclic carbonates using natural amino acids was also investigated by Liu and coworkers (Liu et al., 2008). The biodegradable polymers with incorporated amino acids molecules were effectively obtained. The presence of natural amino acids in the polymer

Polyamidoamine (PAMAM) dendrimers are highly hyperbranched synthetic polymers with well-defined structure that allows accurate control of shape, size and functionality of terminal groups (Tomalia et al., 1985). The PAMAM dendrimers have useful applications in pharmaceutical technology e.g. as carriers in drug delivery systems, which can cross cell walls by both paracellular and transcellular pathways (Jevprasesphant et al., 2003). In our laboratory, novel star-shaped biodegradable polyesters were synthesized by ROP of CL using PAMAM dendrimer initiator without any metal catalysts (Oledzka et al., 2011). The nonlinear structure as well as physicochemical properties of the obtained biodegradable polymers were confirmed by nuclear magnetic resonance, gel permeation chromatography,

**3.3 Natural co-initiators of ring-opening polymerization of cyclic esters, ether-esters**

Metal catalyzed ROP of cyclic esters, ether-esters or carbonates has become the object of intensive studies with respect to their practical applications in the synthesis of biomaterials.

synthesized with high yield (even ca. 100 % in some cases).

Fig. 6. ROP of cyclic esters using natural amino acids as initiators

chain was proved by nuclear magnetic resonance spectroscopy.

thermal gravimetric analysis and differential scanning calorymetry.

observed over time.

**and carbonates**

Tin(II) 2-ethylhexanoate (SnOct2) is commonly used as a commercial catalyst for the ROP of cyclic monomers. It is effective, relatively cheap, non-toxic, soluble in the most commonly used organic solvents (Labet & Thielemans; 2009). SnOct2 is considered to have a toxicity much lower than other metal compounds, and it is allowed to be used as a food additive in a number of countries.

SnOct2 must be used together with a nucleophilic compound (generally an alcohol) to initiate the reaction if a controlled synthesis of the polymer is to be obtained. The main drawback of SnOct2 is that it requires high temperature, which leads inter- and intramolecular esterification (Labet & Thielemans; 2009).

According to Kowalski's hypothesis, the first step of the polymerization consists of the production of the active species by reacting the alcohol with the catalyst. The more alcohol is added, the more the equilibrium is displaced towards the right and the more active species are created. With increasing carboxylic acid concentration, the equilibrium shifts to the left and less active species are present in the medium (Scheme 3) (Kowalski et al., 1998). Mechanism of CL and LA polymerization initiated with SnOct2/C4H9NH2 system has also been described (Duda et al., 2005).

Scheme 3. The formation of active centres in the reaction of Sn(Oct)2 with alcohol (co-initiator)

SnOct2 has also been combined with ureidopyrimidinone-alcohol (UPy) compounds. Using the good soluble alcohols, bearing a 1-ethylpentyl moiety, the ROP was significantly more controlled (Celiz & Scherman, 2008).

Sobczak and Kolodziejski have studied SnOct2/L-carnitine (CA) catalytic system. CA is a hydrophilic amino acid derivative, naturally occurring in human cell. Low-molecular weight PCL, PLA and copolymers of CL and *rac*-LA were obtained by the ROP of cyclic esters in the presence of SnOct2/CA. The molecular mass values averaged over the obtained polyesters were roughly in agreement with the theoretical molecular weights calculated from the feed ratio of the cyclic esters to CA (Sobczak & Kolodziejski, 2009).

Zhang and coworkers used cholesterol (CHL) as an initiator and SnOct2 as a catalyst of ROP of CL (Zhang et al., 2005). The polymerization was carried out under rigorously anhydrous conditions. The optimized ring-opening polymerization conditions have been identified to be 8 h at 140 ºC. The molecular weight of CHL-PCL has increased with decreasing cholesterol/CL feed ratio. Incorporation of the cholesteryl moiety into polymer chain has leaded to a slower enzymatic degradation rate. Whereas, Cai and coworkers utilized PAMAM dendrimer as initiator of ROP of LLA (Cai et al., 2003). The star-shaped biodegradable polymers with the average molecular weight about 70000 Da were successfully obtained in that work. The authors also found that the synthesized polymers showed a faster degradation rate than linear homopolymer because of its shortened polymer chains.

### **4. Conclusions**

The pharmaceutical technology is one of the most important fields of using of polymers. From this review, it is clear that macromolecules have been extremely active research area over the last years. In addition is worth to note, that the progress of modern pharmaceutical technology is not feasible without utilization of natural and synthetic polymers. The discovering of new drug forms, e.g.: new therapeutic systems and macromolecular prodgrugs is simply demanded by the market and industry presently. The elaboration of new medical and pharmaceutical specimens will also require intensive investigations in chemistry and biomedical polymer areas.

As is also evident from this discussion, the spectacular improvement has been achieved with natural compounds applied as initiators, catalysts, organocatalysts or co-initiators of polymerization of cyclic esters, ether-esters and carbonates. The utilized compounds are primarily friendly for environment, safe, non-toxic and irreplaceable for the synthesis of polymers for the pharmaceutical applications. Promising avenues of research have also emerged for the enzymatic approach. Increasing interest has also been dedicated to the polymers containing natural compounds in macromolecules that have been incorporated into though the polymerization process. Clearly, the future development of biodegradable and bioresorbable polymers will be based on discovering macromolecules with not only appropriate chemical, physical and mechanical properties but also suitable biological properties.

#### **5. Acknowledgment**

We would like to thank Professor M. Naruszewicz for financial support.

#### **6. References**

The pharmaceutical technology is one of the most important fields of using of polymers. From this review, it is clear that macromolecules have been extremely active research area over the last years. In addition is worth to note, that the progress of modern pharmaceutical technology is not feasible without utilization of natural and synthetic polymers. The discovering of new drug forms, e.g.: new therapeutic systems and macromolecular prodgrugs is simply demanded by the market and industry presently. The elaboration of new medical and pharmaceutical specimens will also require intensive investigations in

As is also evident from this discussion, the spectacular improvement has been achieved with natural compounds applied as initiators, catalysts, organocatalysts or co-initiators of polymerization of cyclic esters, ether-esters and carbonates. The utilized compounds are primarily friendly for environment, safe, non-toxic and irreplaceable for the synthesis of polymers for the pharmaceutical applications. Promising avenues of research have also emerged for the enzymatic approach. Increasing interest has also been dedicated to the polymers containing natural compounds in macromolecules that have been incorporated into though the polymerization process. Clearly, the future development of biodegradable and bioresorbable polymers will be based on discovering macromolecules with not only appropriate chemical, physical and mechanical properties but also suitable biological

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## **Translating 2A Research into Practice**

### Garry A. Luke

*University of St Andrews, Scotland UK*

#### **1. Introduction**

160 Innovations in Biotechnology

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functionalization of poly(ε-caprolactone) by cholesteryl moiety. *Journal of Biomaterials Science*, *Polymer Edition,* Vol.16, No.9, (May 2005), pp. 1095-1108, ISSN

> Viruses have evolved a number of unconventional translation strategies to amplify the coding potential of their condensed genetic information. Leaky stop codons may be readthrough to produce either the predicted translation product, or at a very low level an extended "read-through" protein. Overlapping (*e.g.* –UAAUG-; -UGAUG-; AUGA-), or highly proximal stop/start codons may give rise to termination accompanied by a low level of re-initiation. There are a number of cases where a single mRNA is translated into more than one protein by recoding, where the rules for decoding are altered through specific sites and signals in the mRNA such as frameshifting and readthrough. Ribosomal "skipping", first identified in the foot-and-mouth disease virus (FMDV), represents yet another translational trick to deliver multiple gene products from limited primary sequence. Briefly, when a ribosome encounters 2A within an open reading frame (ORF), the synthesis of a specific peptide bond is "skipped". The process gives rise to two alternative outcomes: either (i) translation terminates at the end of 2A, or (ii) translation of the downstream sequence occurs. In this manner discrete translation products can be synthesized from a single ORF (for in-depth reviews of recoding see Atkins & Gesteland, 2010).

> 2A and "2A-like" sequences have been thoroughly studied in the last 25 years. These results, as well as our current understanding of the underlying mechanism, are summarized in the first section of this review. In the next section, important considerations in the design of 2A peptide-linked vectors are discussed. The 2A peptide system has worked in all eukaryotic systems tested and has been used with some spectacular successes in a variety of biotechnology applications. In the final section we provide an overview of the literature highlighting some of these successes.

#### **2. Basic research**

#### **2.1 The fmdv genome**

The FMDV genome organization is similar to that of other picornaviruses, comprising a large single ORF flanked by highly structured 5' and 3' untranslated regions (UTRs) (Fig. 1). The 5' UTR, of approximately 1,300 nucleotides (nt) contains sequence elements controlling the replication of viral RNA, packaging of RNA into capsids, and translation of the viral polyprotein. Preceding the ORF is a type II internal ribosome entry site (IRES), crucial for the cap-independent initiation of translation (for reviews see Jackson et al., 1990; Martĩnez-Salas & Ryan, 2010). The 3' UTR is about 90 nt long and is thought to contain cis-acting elements

Fig. 1. The FMDV Genome. The FMDV genome is organized like a cellular mRNA: a 5' untranslated region (5'UTR), a single open reading frame (ORF), a 3' untranslated region (3'UTR) and a poly(A) tail. The polyprotein (~2,300aa) undergoes three "primary", cotranslational cleavages; Lpro cleaves at its own C-terminus, 2A mediates "cleavage" at its own C-terminus and 3Cpro cleaves between [2BC] and 3A. The 2A oligopeptide is only 18aa long, mediating a "cleavage" by a translational effect "ribosome skipping".

required for efficient genome replication (Agol et al., 1999). Moreover, the 3' end of mRNA has also turned out to be surprisingly important in regulating translation (Wells et al., 1998). The ORF encodes a large protein precursor (polyprotein) which can be divided into three regions, designated P1, P2, and P3. These correspond to the N-terminal capsid protein precursor (P1, containing four capsid proteins 1A-1D), the middle of the polyprotein containing three of the nonstructural proteins (P2, the three proteins 2A-2C), and the most Cterminal segment of the polyprotein containing four non-structural proteins (P3, proteins 3A-3D) (Palmenberg, 1987). The full-length translation product is never observed within infected cells due to co-translational, intramolecular, cleavages mediated by Lpro, 2A and 3Cpro domains within the polyprotein (for reviews see, Belsham, 2005; Ryan et al., 2004). Besides releasing itself from the polyprotein, Lpro, in common with 2Apro of the entero- and rhinoviruses, also cleaves the translation initiation factor eIF4G (Glaser & Skern, 2000). This results in the inactivation of cap-dependent translation leading to the shutoff of cellular protein synthesis. The 2A oligopeptide is responsible for the primary cleavage which separates the region comprising the capsid proteins from domains downstream of 2A concerned with the replication of the virus (Ryan et al., 1991; Ryan & Drew, 1994). All picornaviruses encode 3Cpro, which carries out a primary cleavage between 2C and 3A and secondary processing of the [P1- 2A], [2BC] and P3 precursors. In FMDV, 3Cpro also cleaves between 2B and 2C (for review see

Fig. 1. The FMDV Genome. The FMDV genome is organized like a cellular mRNA: a 5' untranslated region (5'UTR), a single open reading frame (ORF), a 3' untranslated region (3'UTR) and a poly(A) tail. The polyprotein (~2,300aa) undergoes three "primary", cotranslational cleavages; Lpro cleaves at its own C-terminus, 2A mediates "cleavage" at its own C-terminus and 3Cpro cleaves between [2BC] and 3A. The 2A oligopeptide is only 18aa

required for efficient genome replication (Agol et al., 1999). Moreover, the 3' end of mRNA has also turned out to be surprisingly important in regulating translation (Wells et al., 1998). The ORF encodes a large protein precursor (polyprotein) which can be divided into three regions, designated P1, P2, and P3. These correspond to the N-terminal capsid protein precursor (P1, containing four capsid proteins 1A-1D), the middle of the polyprotein containing three of the nonstructural proteins (P2, the three proteins 2A-2C), and the most Cterminal segment of the polyprotein containing four non-structural proteins (P3, proteins 3A-3D) (Palmenberg, 1987). The full-length translation product is never observed within infected cells due to co-translational, intramolecular, cleavages mediated by Lpro, 2A and 3Cpro domains within the polyprotein (for reviews see, Belsham, 2005; Ryan et al., 2004). Besides releasing itself from the polyprotein, Lpro, in common with 2Apro of the entero- and rhinoviruses, also cleaves the translation initiation factor eIF4G (Glaser & Skern, 2000). This results in the inactivation of cap-dependent translation leading to the shutoff of cellular protein synthesis. The 2A oligopeptide is responsible for the primary cleavage which separates the region comprising the capsid proteins from domains downstream of 2A concerned with the replication of the virus (Ryan et al., 1991; Ryan & Drew, 1994). All picornaviruses encode 3Cpro, which carries out a primary cleavage between 2C and 3A and secondary processing of the [P1- 2A], [2BC] and P3 precursors. In FMDV, 3Cpro also cleaves between 2B and 2C (for review see

long, mediating a "cleavage" by a translational effect "ribosome skipping".

Martĩnez-Salas & Ryan, 2010). Aside from the processing sites within the viral polyprotein itself, the enzyme also modifies host cell proteins (Belsham et al., 2000; Li et al., 2001).

Fig. 2. Analysis of 2A-mediated "cleavage". Artificial polyprotein cDNA constructs comprising the reporter proteins green fluorescent protein (GFP) and β-glucuronidase (GUS) (left panel). SDS-PAGE of radiolabeled *in vitro* translation products (right panel). The control pGFPGUS construct produces only a single translation product – the [GFP-GUS] fusion protein. The translation profile from the pGFP2AGUS construct shows 3 major products: uncleaved [GFP2AGUS] and the cleavage products [GFP2A] and [GUS]. The profile from pGUS2AGFP also shows 3 major products: uncleaved [GUS2AGFP] and the cleavage products [GUS-2A] and [GFP]. The cleavage products upstream of 2A are highlighted in red, showing the molar excess over the downstream products shown in blue.

Secondary 3Cpro cleavage of the [1D2A] precursor protein between 1D and 2A shows the FMDV 2A segment is only 18aa long (-LLNFDLLKLAGDVESNPG-) (Belsham, 1993). Analysis of recombinant polyproteins and artificial polyprotein systems in which 2A was inserted between two reporter proteins showed that 2A alone, plus the N-terminal proline of protein 2B, was sufficient to mediate a highly efficient co-translational "cleavage" at the C-terminus of 2A (Ryan & Drew, 1994; Ryan et al., 1991; de Felipe et al., 2003). Translation *in vitro*, together with careful quantification of the products (Fig.2), provided the major finding that a molar excess of protein encoded upstream of 2A accumulated over that downstream – an observation at variance with proteolytic processing (Ryan et al., 1989; Donnelly et al., 2001a).

Extensive protein degradation studies, examining the effects of non-specific premature termination of transcription/translation, have shown that none of these effects account for this imbalance (Ryan et al., 1999). Addition of puromycin at low concentration to translation reactions programmed with mRNA encoding a 2A containing reporter yields significant product with a size corresponding to the protein up to the 2A site, indicating a pause in translation at this position (Donnelly et al., 2001a). Employing a "toe-printing" approach, Doronina and colleagues confirmed that ribosomes pause at the end of the 2A coding sequence (-NPGP-), with glycine and proline in the P- and A- sites, respectively (Doronina et al., 2008b). This front end loading was due to different rates of biosynthesis of each portion of the ORF and constitutes a novel type of recoding (Baranov et al., 2002; Brown & Ryan, 2010).

#### **2.2 The cleavage mechanism**

The 2A region of the FMDV encodes a sequence that mediates self-processing by a novel translational effect variously referred to as "ribosome skipping" (Ryan et al., 1999), "stop-go" (Atkins et al., 2007) and "stop carry-on" translation (Doronina et al., 2008a). 2A-mediated cleavage occurs between the C-terminal glycine and the proline of the downstream protein 2B (-LLNFDLLKLAGDVESNPGP-). The upstream protein contains a short 2A peptide Cterminal fusion, whereas the downstream protein includes a single proline residue on its Nterminus (Ryan and Drew, 1994; Ryan et al., 1991). The translational model of 2A cleavage activity posited is shown in Figure 3. Briefly, the nascent 2A peptide interacts with the exit pore of the ribosome such that the C-terminal portion (-ESNPGP-) is sterically constrained within the peptidyl transferase centre of the ribosome. This inhibits nucleophilic attack of the ester linkage between 2A and tRNAgly by prolyl-tRNA in the A site - effectively stalling, or pausing, translation (Ryan et al., 1999; Donnelly et al., 2001a). It has been shown that this block is relieved by the action of translation release factors eRF1 and eRF3, hydrolysing the ester linkage and releasing the nascent protein (Doronina et al., 2008a & b). Thus two major outcomes are possible; either translation terminates at this point, or, translation effectively 'reinitiates' to synthesize the downstream sequences. The latter case would entail; (a) egress of eRF1/3 from the A site, (b) ingress of prolyl-tRNA into the A site, (c) translocation of prolyltRNA to the P site and (d) entry of the next aminoacyl-tRNA (for in-depth reviews of the model see Ryan et al., 2002; Martĩnez-Salas & Ryan, 2010; Brown & Ryan, 2010).

#### **2.3 The occurrence of 2A and 2A-like sequences**

Examining other picornavirus genome sequences showed the DxExNPGP motif to be present in several genera of the *Picornaviridae*: aphtho- cardio-, tescho-, erbo- and certain parechoviruses. Although cardioviruses have much longer natural 2A segments (133 to 143 amino acids) than aphthoviruses, work with *Encephalomyocarditis virus* (EMCV) and *Theiler's murine encephalitis virus* (TMEV) has shown that most of the additional 2A protein is dispensable for primary cleavage activity (Hahn & Palmenberg, 1996; Donnelly et al., 1997). Probing databases for the presence of the motif showed that "2A-like" sequences were also present in a range of non-picornavirus systems. These include a wide range of insect positive-strand RNA viruses belonging to the *Dicistroviridae* and *Tetraviridae* families and the unassigned Iflavirus genus and double-stranded RNA viruses of the *Reoviridae* (insect Cypoviruses and mammalian type C rotaviruses) (Hahn & Palmenberg, 1996; Donnelly et al., 2001b). They are also found in four nonsegmented dsRNA viruses of the *Totiviridae* (Isawa et al., 2011). Analysis of the translation products showed that in all cases these 2As had "cleavage" activity (Luke et al., 2008).

imbalance (Ryan et al., 1999). Addition of puromycin at low concentration to translation reactions programmed with mRNA encoding a 2A containing reporter yields significant product with a size corresponding to the protein up to the 2A site, indicating a pause in translation at this position (Donnelly et al., 2001a). Employing a "toe-printing" approach, Doronina and colleagues confirmed that ribosomes pause at the end of the 2A coding sequence (-NPGP-), with glycine and proline in the P- and A- sites, respectively (Doronina et al., 2008b). This front end loading was due to different rates of biosynthesis of each portion of the ORF and constitutes a novel type of recoding (Baranov et al., 2002; Brown & Ryan, 2010).

The 2A region of the FMDV encodes a sequence that mediates self-processing by a novel translational effect variously referred to as "ribosome skipping" (Ryan et al., 1999), "stop-go" (Atkins et al., 2007) and "stop carry-on" translation (Doronina et al., 2008a). 2A-mediated cleavage occurs between the C-terminal glycine and the proline of the downstream protein 2B (-LLNFDLLKLAGDVESNPGP-). The upstream protein contains a short 2A peptide Cterminal fusion, whereas the downstream protein includes a single proline residue on its Nterminus (Ryan and Drew, 1994; Ryan et al., 1991). The translational model of 2A cleavage activity posited is shown in Figure 3. Briefly, the nascent 2A peptide interacts with the exit pore of the ribosome such that the C-terminal portion (-ESNPGP-) is sterically constrained within the peptidyl transferase centre of the ribosome. This inhibits nucleophilic attack of the ester linkage between 2A and tRNAgly by prolyl-tRNA in the A site - effectively stalling, or pausing, translation (Ryan et al., 1999; Donnelly et al., 2001a). It has been shown that this block is relieved by the action of translation release factors eRF1 and eRF3, hydrolysing the ester linkage and releasing the nascent protein (Doronina et al., 2008a & b). Thus two major outcomes are possible; either translation terminates at this point, or, translation effectively 'reinitiates' to synthesize the downstream sequences. The latter case would entail; (a) egress of eRF1/3 from the A site, (b) ingress of prolyl-tRNA into the A site, (c) translocation of prolyltRNA to the P site and (d) entry of the next aminoacyl-tRNA (for in-depth reviews of the

model see Ryan et al., 2002; Martĩnez-Salas & Ryan, 2010; Brown & Ryan, 2010).

Examining other picornavirus genome sequences showed the DxExNPGP motif to be present in several genera of the *Picornaviridae*: aphtho- cardio-, tescho-, erbo- and certain parechoviruses. Although cardioviruses have much longer natural 2A segments (133 to 143 amino acids) than aphthoviruses, work with *Encephalomyocarditis virus* (EMCV) and *Theiler's murine encephalitis virus* (TMEV) has shown that most of the additional 2A protein is dispensable for primary cleavage activity (Hahn & Palmenberg, 1996; Donnelly et al., 1997). Probing databases for the presence of the motif showed that "2A-like" sequences were also present in a range of non-picornavirus systems. These include a wide range of insect positive-strand RNA viruses belonging to the *Dicistroviridae* and *Tetraviridae* families and the unassigned Iflavirus genus and double-stranded RNA viruses of the *Reoviridae* (insect Cypoviruses and mammalian type C rotaviruses) (Hahn & Palmenberg, 1996; Donnelly et al., 2001b). They are also found in four nonsegmented dsRNA viruses of the *Totiviridae* (Isawa et al., 2011). Analysis of the translation products showed that in all cases these 2As

**2.3 The occurrence of 2A and 2A-like sequences**

had "cleavage" activity (Luke et al., 2008).

**2.2 The cleavage mechanism**

Fig. 3. Schematic representation of the translational model of 2A-mediated "cleavage".

Along with the RNA virus 2As, active 2A-like sequences were also detected in the N-terminal region of the ORFs of non-LTR retrotransposons of *Trypanosoma cruzi* and *T.brucei* – L1Tc and igni, respectively (Donnelly et al., 2001b; Heras et al., 2006). Recently we identified a range of 2As in the purple sea urchin *Stronglocentrotus purpuratus*, then demonstrated their cleavage activities (unpublished data). In this case, 2A-like sequences appear in (i) several copies of non-LTR-retroelements (like trypanosomes) and (ii) the N-terminus of nucleotide binding oligomerization domain (NOD)-like, or CATERPILLER proteins (cited in Brown & Ryan, 2010). It appears, therefore, that this method of controlling protein biogenesis is not confined to viruses or genomic sequences comprising insertion(s) of virus-related sequences (retroelements). 2A and "2A-like" sequences have been shown to function in cells from a wide variety of eukaryotes, ranging from yeast (de Felipe et al., 2003) to plants (Halpin et al., 1999) to insects (Roosien et al., 1990) to mammals (Ryan & Drew, 1994). The only requirement for 2A peptide-based cleavage appears to be translation by 80S ribosomes. The reported proteolysis activity of 1D-2A in *E.coli* cells (Dechamma et al., 2008) was not detected in equivalent constructions in our laboratory showing "cleavage" specificity for eukaryotic systems alone (Donnelly et al., 1997). Although the FMDV 2A sequence (hereafter referred to as "F2A") has been the most widely used, biotechnologists should be aware that many 2A-like sequences have been utilized successfully, including equine rhinitis A virus (ERAV, "E2A"), porcine teschovirus-1 (PTV-1, "P2A") and *Thosea asigna* virus (TaV, "T2A") (Szymczak et al., 2004; Arnold et al., 2004; Osborn et al., 2005; Szymczak & Vignali, 2005; Huang et al., 2006; Scholten et al., 2006; Hart et al., 2008; Sommer et al., 2008; Yang et al., 2008).

#### **3. General considerations when using 2A peptide sequences**

#### **3.1 Expression of multiple genes**

Conventional approaches for the production of multicistronic vectors include the use of IRES elements, multiple promoters, fusion proteins, *etc* (for a review see de Felipe, 2002). Adverse side-effects with multiple promoters on viral vectors include interference between promoters, promoter suppression and rearrangement (Cullen et al., 1984; Emerman & Temin, 1986). IRESes provided the first method of creating eukaryotic polycistronic mRNAs. The internal ribosome entry site serves as a launching pad for internal initiation of translation, allowing expression of two or more genes from a single transcript (for review see Komar & Hatzoglou, 2005). Since genes are under the control of the same promoter and integrated into the same place within the genome, transgenes expressed in this way are coordinately regulated. In bicistronic systems, detection of the product encoded by the second cistron is evidence that the first cistron is also being expressed. This approach has been used successfully in gene therapy research in animal systems, and IRESes from different viruses have been tested and shown to function in plant systems (Urwin et al., 2000 & 2002; Dorokhov et al., 2002; Jaag et al., 2003; Bouabe et al., 2008).

On the other hand, there are a couple of limitations using IRES elements. Firstly, the IRES is a relatively large sequence (~500bp) that can cause problems in packaging, especially for size-restricted viral and nonviral vectors. For instance, retro- and lentiviral vectors possess packaging capacities of 8kb and adeno-associated viruses can accommodate <5kb (Thomas et al., 2003). Secondly, expression of the downstream gene can be as much as 10 fold lower than the upstream gene (Mizuguchi et al. 2000; Flasshove et al., 2000; Hasegawa et al., 2007; Ha et al., 2010). In some instances, this can be useful for expressing fluorescent markers or conferring drug resistance during selection (Ngoi et al., 2004). Nevertheless, the obvious advantages of using the 2A sequence vis-á-vis the IRES are its smaller size (~60-70bp) and the stoichiometric production of both upstream and downstream protein products as measured by: i) chloramphenicol acetyltransferase (CAT) and -glucuronidase (GUS) enzyme activity (Halpin et al., 1999); ii) cell free translation *in vitro* and Western blot (Ryan & Drew, 1994; Donnelly et al., 2001a & b; de Felipe et al., 2003; Torres et al., 2010)*;* iii) GFP/FACS with antibiotic resistance (Lorens et al., 2004); iv) co-fluorescence reporting (de Felipe & Ryan, 2004; Samalova et al., 2006); v) fluorescence resonance energy transfer (FRET) analysis (Szymczak et al., 2004) and vi) protein segregation in genetically engineered animals (Provost et al., 2007; Trichas et al., 2008). Further, if multiple gene expression is required, different members of the 2A peptide family can be selected to disrupt sequence homology to help maintain foreign gene insert stability.

#### **3.2 Subcellular targeting of proteins from a 2A polyprotein**

A merit of this expression strategy is that individual components of the 2A-polyprotein can be targeted to a range of different sub-cellular sites using both co- and post-translational signal sequences (El Amrani et al., 2004; Lorens et al., 2004; Szymczak et al., 2004). We discovered, however, a major problem with co-expression of some proteins targeted to, or passing through, the mammalian endoplasmic reticulum (ER). When a 2A-based polyprotein comprising an upstream protein bearing an N-terminal signal sequence was followed by a protein lacking any signal sequence, both proteins were translocated into the

Conventional approaches for the production of multicistronic vectors include the use of IRES elements, multiple promoters, fusion proteins, *etc* (for a review see de Felipe, 2002). Adverse side-effects with multiple promoters on viral vectors include interference between promoters, promoter suppression and rearrangement (Cullen et al., 1984; Emerman & Temin, 1986). IRESes provided the first method of creating eukaryotic polycistronic mRNAs. The internal ribosome entry site serves as a launching pad for internal initiation of translation, allowing expression of two or more genes from a single transcript (for review see Komar & Hatzoglou, 2005). Since genes are under the control of the same promoter and integrated into the same place within the genome, transgenes expressed in this way are coordinately regulated. In bicistronic systems, detection of the product encoded by the second cistron is evidence that the first cistron is also being expressed. This approach has been used successfully in gene therapy research in animal systems, and IRESes from different viruses have been tested and shown to function in plant systems (Urwin et al., 2000 & 2002;

On the other hand, there are a couple of limitations using IRES elements. Firstly, the IRES is a relatively large sequence (~500bp) that can cause problems in packaging, especially for size-restricted viral and nonviral vectors. For instance, retro- and lentiviral vectors possess packaging capacities of 8kb and adeno-associated viruses can accommodate <5kb (Thomas et al., 2003). Secondly, expression of the downstream gene can be as much as 10 fold lower than the upstream gene (Mizuguchi et al. 2000; Flasshove et al., 2000; Hasegawa et al., 2007; Ha et al., 2010). In some instances, this can be useful for expressing fluorescent markers or conferring drug resistance during selection (Ngoi et al., 2004). Nevertheless, the obvious advantages of using the 2A sequence vis-á-vis the IRES are its smaller size (~60-70bp) and the stoichiometric production of both upstream and downstream protein products as measured by: i) chloramphenicol acetyltransferase (CAT) and -glucuronidase (GUS) enzyme activity (Halpin et al., 1999); ii) cell free translation *in vitro* and Western blot (Ryan & Drew, 1994; Donnelly et al., 2001a & b; de Felipe et al., 2003; Torres et al., 2010)*;* iii) GFP/FACS with antibiotic resistance (Lorens et al., 2004); iv) co-fluorescence reporting (de Felipe & Ryan, 2004; Samalova et al., 2006); v) fluorescence resonance energy transfer (FRET) analysis (Szymczak et al., 2004) and vi) protein segregation in genetically engineered animals (Provost et al., 2007; Trichas et al., 2008). Further, if multiple gene expression is required, different members of the 2A peptide family can be selected to disrupt sequence

A merit of this expression strategy is that individual components of the 2A-polyprotein can be targeted to a range of different sub-cellular sites using both co- and post-translational signal sequences (El Amrani et al., 2004; Lorens et al., 2004; Szymczak et al., 2004). We discovered, however, a major problem with co-expression of some proteins targeted to, or passing through, the mammalian endoplasmic reticulum (ER). When a 2A-based polyprotein comprising an upstream protein bearing an N-terminal signal sequence was followed by a protein lacking any signal sequence, both proteins were translocated into the

**3. General considerations when using 2A peptide sequences**

Dorokhov et al., 2002; Jaag et al., 2003; Bouabe et al., 2008).

homology to help maintain foreign gene insert stability.

**3.2 Subcellular targeting of proteins from a 2A polyprotein**

**3.1 Expression of multiple genes**

ER (de Felipe & Ryan, 2004). We have identified the source of this problem - the "slipstreaming" effect was due to inhibition of the 2A reaction (formation of fusion protein) by the C-terminal region (immediately upstream of 2A) of some proteins when translocated into the ER – and suggest possible solutions (de Felipe et al., 2010).

The residues that influence cleavage are predicted to reside within the translocon; this length may allow interactions between the nascent peptide and the ribosome that lead to inhibition of the 2A reaction (Ménétret et al., 2000; Beckmann et al., 2001; de Felipe et al., 2010). Solutions to the problem include the use of longer versions of 2A with extra sequences derived from the capsid protein ("1D") (Ryan et al., 1991; Groot Bramel-Verheije et al., 2000; Donnelly et al., 2001b; Klump et al., 2001). Specifically, N-terminal extension of 2A by 5aa of 1D improved "cleavage", but extension by 14aa of 1D or longer (21 and 39aa) produced complete "cleavage" and an equal stoichiometry of the up- and downstream translation products (Donnelly et al., 2001b, see fig. 4). These observations are consistent with our model in which 2A activity is a product of it's interaction with the exit tunnel of the ribosome which is thought to accommodate 30-40aa (Hardesty & Kramer, 2001). Further,

Fig. 4. Translation *in vitro*. Translation products derived from constructs encoding the wildtype 2A sequence are shown together with products derived from constructs encoding Nterminally extended forms of 2A.

the order in which the genes are expressed within the vector needs to be considered. By swapping the order of proteins in several artificial polyproteins the stoichiometry was affected by the gene upstream of 2A (Ma & Mitra, 2002; Lengler et al., 2005; Chinnasamy et al., 2006; Rothwell et al., 2010). Cleavage activity was independent of the immediate downstream sequence (Ryan et al., 1991; Ma & Mitra., 2002). A number of studies show that cleavage efficiency is improved by incorporation of a flexible Gly-Ser-Gly or Ser-Gly-Ser-Gly linker sequence between the upstream protein and the 2A peptide (Lorens et al., 2004; Szymczak et al., 2004; Holst et al., 2006a & b; Provost et al., 2007; Wargo et al., 2009). A noteworthy caveat to attach to this review is that Yan and colleagues argue slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires a signal sequence for secretory or membrane-anchored expression (Yan et al., 2010).

#### **3.3 The unwanted tags**

Cleavage occurs at the end of the 2A peptide sequence, therefore most of the 2A remains attached to the C-terminus of the upstream protein. This may affect the activity of some proteins (*e.g.* if their function is affected by the addition of other tags such as Myc, His, *etc*). In the case of proteins translocated into the ER, a strategy was adopted to include a furin proteinase cleavage site (-RAKR-) between the upstream protein and 2A (Fang et al., 2005). Furin is a cellular endoprotease localized on the trans-Golgi networks of virtually all cell types (Steiner, 1998). Upon entering the lumen of the ER, 2A was trimmed away from the upstream protein (in this case antibody heavy chain), leaving only a 2aa C-terminal extension (-RA). In a follow-on study, the use of alternative furin cleavage sequences consisting of only basic amino acids, which can be efficiently cut by carboxypeptidases (- RRRR-, -RKRR-, -RRKR-), resulted in the expression of antibodies with no residual amino acids (Fang et al., 2007). Proteins expressed in plants could have their 2A extensions removed by endogenous proteinases acting on similar hybrid linker peptides. A polyprotein precursor consisting of two different marker proteins connected by a linker peptide of Impatiens balsamina (-SNAADEVAT-) followed by F2A was successfully processed in *Arabidopsis thaliana* (François et al., 2002; François et al., 2004).

For biomedical applications using 2A, a concern stems from the addition of 2A-derived sequences to the upstream protein - this protein may act as a carrier to stimulate an anti-2A immune response. Any potential carrier-effects could be abolished by removal of 2A. It should be noted that this "unwanted" tag does confer two advantages. First, antibodies to the 2A-peptide have been generated, allowing detection and/or immunoprecipitation of "upstream" protein products derived from 2A-containing transgenes (de Felipe et al, 2006 & 2010). Second, a shift in protein size is observed in 2A-tagged proteins which can be useful if mutant and endogenous proteins are co-expressed and need to be identified (Szmczak et al., 2004; de Felipe et al., 2010). To our knowledge, the presence of a proline attached to the amino-terminus of the second protein, as a relic of the 2A self-cleaving process, does not interfere significantly with activity and trafficking; it does, however, confer high protein stability (Varshavsky, 1992).

#### **4. Translational studies**

#### **4.1 Gene expression** *in planta*

Currently, there are several options available for the introduction of multiple transgenes *in planta*. The different methods include sexual crossing, re-transformation, single-plasmid or

et al., 2006; Rothwell et al., 2010). Cleavage activity was independent of the immediate downstream sequence (Ryan et al., 1991; Ma & Mitra., 2002). A number of studies show that cleavage efficiency is improved by incorporation of a flexible Gly-Ser-Gly or Ser-Gly-Ser-Gly linker sequence between the upstream protein and the 2A peptide (Lorens et al., 2004; Szymczak et al., 2004; Holst et al., 2006a & b; Provost et al., 2007; Wargo et al., 2009). A noteworthy caveat to attach to this review is that Yan and colleagues argue slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires a signal sequence for secretory or membrane-anchored

Cleavage occurs at the end of the 2A peptide sequence, therefore most of the 2A remains attached to the C-terminus of the upstream protein. This may affect the activity of some proteins (*e.g.* if their function is affected by the addition of other tags such as Myc, His, *etc*). In the case of proteins translocated into the ER, a strategy was adopted to include a furin proteinase cleavage site (-RAKR-) between the upstream protein and 2A (Fang et al., 2005). Furin is a cellular endoprotease localized on the trans-Golgi networks of virtually all cell types (Steiner, 1998). Upon entering the lumen of the ER, 2A was trimmed away from the upstream protein (in this case antibody heavy chain), leaving only a 2aa C-terminal extension (-RA). In a follow-on study, the use of alternative furin cleavage sequences consisting of only basic amino acids, which can be efficiently cut by carboxypeptidases (- RRRR-, -RKRR-, -RRKR-), resulted in the expression of antibodies with no residual amino acids (Fang et al., 2007). Proteins expressed in plants could have their 2A extensions removed by endogenous proteinases acting on similar hybrid linker peptides. A polyprotein precursor consisting of two different marker proteins connected by a linker peptide of Impatiens balsamina (-SNAADEVAT-) followed by F2A was successfully processed in

For biomedical applications using 2A, a concern stems from the addition of 2A-derived sequences to the upstream protein - this protein may act as a carrier to stimulate an anti-2A immune response. Any potential carrier-effects could be abolished by removal of 2A. It should be noted that this "unwanted" tag does confer two advantages. First, antibodies to the 2A-peptide have been generated, allowing detection and/or immunoprecipitation of "upstream" protein products derived from 2A-containing transgenes (de Felipe et al, 2006 & 2010). Second, a shift in protein size is observed in 2A-tagged proteins which can be useful if mutant and endogenous proteins are co-expressed and need to be identified (Szmczak et al., 2004; de Felipe et al., 2010). To our knowledge, the presence of a proline attached to the amino-terminus of the second protein, as a relic of the 2A self-cleaving process, does not interfere significantly with activity and trafficking; it does, however, confer high protein

Currently, there are several options available for the introduction of multiple transgenes *in planta*.

**4. Translational studies 4.1 Gene expression** *in planta*

**3.3 The unwanted tags**

multiple-plasmid co-transformation, and IRES-based transformation. The pros and cons of each have been reviewed previously (François et al., 2002; Halpin, 2005; Luke et al., 2006 & 2010). However, these procedures all suffer from a lack of coordinated expression of the different transgenes. As an alternative, the coding sequences of the genes of interest can be linked *via* 2A in a single transcription unit (Halpin et al., 1999; Ma & Mitra, 2002). The first types of genetically modified organisms created using 2A to co-express multiple proteins were plants, initially as a research tool, but also to improve drought-resistance (Kwon et al., 2004); disease-resistance (François et al., 2004; Geu-Flores et al., 2009) and nutritional qualities (Randall et al., 2004).

Metabolic and combinatorial engineering of carotenoid biosynthetic pathways in plants, including those synthesizing important industrial and pharmaceutical products, provide excellent examples of the utility of this approach (Ralley et al., 2004; Ha et al., 2010). Carotenoids have attracted interest not only as a source of pigmentation but also for their beneficial effects on human health. One of the most widely known carotenoids is -carotene, which serves as a dietary precursor of vitamin A. In developing countries, where vitamin A deficiency prevails, a promising intervention to existing strategies is to fortify the major staple food, rice, with provitamin A. Golden rice (*Oryza sativa*, GR) is the generic name given to genetically modified rice that produces -carotene in the endosperm (Ye et al., 2000, GR1; Paine et al., 2005, GR2). Engineering the provitamin A (-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm requires two carotenoid biosynthetic genes, phytoene synthase (*psy*) and carotene desaturase (*crtl*) (Lu & Li, 2008). As a step towards the coordinate expression of the two genes, *psy* from *Capsicum* and *crtl* from *Pantoea*, were linked *via* synthetic 2A (*psy-F2A-crtl*) or IRES (*psy-IRES-crtl*) sequences and placed under the control of the rice endosperm-specific globulin promoter (Ha et al., 2010). Collectively, the results demonstrated that the 2A construct performed better than the IRES construct in terms of carotenoid production. In addition, the use of a single promoter (GR1 and 2 require two promoters) reduces the chance of gene silencing and provides more space for transgene stacking.

#### **4.2 Optical imaging of gene expression**

In order to monitor transgene delivery and expression by optical imaging, the coding region is fused to a fluorescent/luminescent reporter. Another approach is to detect the expressed protein through its activity (conversion of a substrate in a fluorescent product as with galactosidase). We (Halpin et al., 1999; Funston et al., 2008; de Felipe et al., 2010) and others (Samalova et al., 2006 & 2008; Hasegawa et al., 2007; Torres et al., 2010) have successfully used the 2A sequence in a number of *in vitro* and *in vivo* heterologous systems to achieve production of various combinations of fluorescent proteins and proteins requiring discrete co- and post-translational subcellular localization. The zebrafish (*Danio rerio*) has proved to be an excellent vertebrate model system for basic and biomedical science and comparative genomics. The lauded advantage of zebrafish embryos being transparent lends itself remarkably well to the use of fluorescence. To demonstrate the utility of the 2A system in zebrafish, reporter constructs employing eGFP and mCherry separated by the P2A sequence were designed to segregate fluorescent proteins to distinct cellular locations (Provost et al., 2007). Tissue-specific expression of both fluorophores in stably transformed embryos shows this approach could facilitate continuous expression of multiple proteins products at various stages of development in zebrafish. Likewise, Trichas et al (2008) used a bi-cistronic reporter construct containing a single coding sequence for a membrane localized red fluorescent protein (Myr-TdTomato) and a nuclear localized green fluorescent protein (H2B-GFP) separated by the T2A sequence to test 2A function in transgenic mice. Mutually exclusive localization of TdTomato and EGFP to the membrane and nucleus was observed in cultured cells and endogenous vertebrate cells, consistent with complete 2A-mediated processing. For the transgenic mice produced in this study, targeted expression was apparent in all tissues examined throughout development and into adulthood and remained constant across several generations.

*In vivo* bioluminescent imaging (BLI) allows a low-cost, noninvasive, and real-time analysis of biological processes at the molecular level in living systems. Cao and colleagues used BLI to visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase (Cao et al., 2005). The donor mouse has a transgene comprised of a hybrid CMV--actin promoter, a firefly luciferase gene, a F2A and GFP gene. Isolated haematopoietic stem cells (HSC) from these mice express luciferase at the highest level among different haematopoietic cell types, and all haematopoietic lineages tested (with the exception of erythroblasts and red blood cells) express the reporter gene. As a virtually unlimited source of labelled cells this mouse line represents a valuable resource for stem cell and transplantation studies.

#### **4.3 Immunotherapies**

#### **4.3.1 Cancer immunotherapy using heat shock protein**

In an effort to extend the scope of immunotherapy for the control of advanced ovarian cancer, BLI was used to measure tumour load and distribution in mice vaccinated with irradiated tumour cells secreting heat shock protein 70 (Hsp70) (Chang et al., 2007). Hsps, including Hsp70, are highly effective in potentiating immune responses *via* interaction with several surface receptors on antigen-presenting cells (APCs). Hsp-specific receptors efficiently transport the chaperoned peptide into the major histocompatibility complex (MHC) class 1 cross-presentation pathway leading to recognition and activation of cytotoxic T cells (Udono & Srivastava, 1993; Massa et al., 2004). A retrovirus encoding sHsp70-T2A-GFP was used to introduce the gene for secreted hsp70 directly into mouse ovarian surface epithelial cells (MOSEC) that express luciferase. In summary, the tumour-secreted Hsp70 was capable of generating a potent antigen-specific "cytotoxic" CD8+ T-cell response and CD40 was identified as a likely receptor for Hsp70-mediated cross-presentation.

#### **4.3.2 Immunotherapy using monoclonal antibodies**

Advances in recent years delineating the specific components of the immune system that contribute to immune responsiveness point to an important regulatory role for immunomodulators. Monoclonal antibodies (mAbs) are an important class of therapeutic agents for the treatment of cancer, autoimmune disorders, and infectious diseases. Although

zebrafish, reporter constructs employing eGFP and mCherry separated by the P2A sequence were designed to segregate fluorescent proteins to distinct cellular locations (Provost et al., 2007). Tissue-specific expression of both fluorophores in stably transformed embryos shows this approach could facilitate continuous expression of multiple proteins products at various stages of development in zebrafish. Likewise, Trichas et al (2008) used a bi-cistronic reporter construct containing a single coding sequence for a membrane localized red fluorescent protein (Myr-TdTomato) and a nuclear localized green fluorescent protein (H2B-GFP) separated by the T2A sequence to test 2A function in transgenic mice. Mutually exclusive localization of TdTomato and EGFP to the membrane and nucleus was observed in cultured cells and endogenous vertebrate cells, consistent with complete 2A-mediated processing. For the transgenic mice produced in this study, targeted expression was apparent in all tissues examined throughout development and into adulthood and remained constant

*In vivo* bioluminescent imaging (BLI) allows a low-cost, noninvasive, and real-time analysis of biological processes at the molecular level in living systems. Cao and colleagues used BLI to visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase (Cao et al., 2005). The donor mouse has a transgene comprised of a hybrid CMV--actin promoter, a firefly luciferase gene, a F2A and GFP gene. Isolated haematopoietic stem cells (HSC) from these mice express luciferase at the highest level among different haematopoietic cell types, and all haematopoietic lineages tested (with the exception of erythroblasts and red blood cells) express the reporter gene. As a virtually unlimited source of labelled cells this mouse line represents a valuable

In an effort to extend the scope of immunotherapy for the control of advanced ovarian cancer, BLI was used to measure tumour load and distribution in mice vaccinated with irradiated tumour cells secreting heat shock protein 70 (Hsp70) (Chang et al., 2007). Hsps, including Hsp70, are highly effective in potentiating immune responses *via* interaction with several surface receptors on antigen-presenting cells (APCs). Hsp-specific receptors efficiently transport the chaperoned peptide into the major histocompatibility complex (MHC) class 1 cross-presentation pathway leading to recognition and activation of cytotoxic T cells (Udono & Srivastava, 1993; Massa et al., 2004). A retrovirus encoding sHsp70-T2A-GFP was used to introduce the gene for secreted hsp70 directly into mouse ovarian surface epithelial cells (MOSEC) that express luciferase. In summary, the tumour-secreted Hsp70 was capable of generating a potent antigen-specific "cytotoxic" CD8+ T-cell response and

CD40 was identified as a likely receptor for Hsp70-mediated cross-presentation.

Advances in recent years delineating the specific components of the immune system that contribute to immune responsiveness point to an important regulatory role for immunomodulators. Monoclonal antibodies (mAbs) are an important class of therapeutic agents for the treatment of cancer, autoimmune disorders, and infectious diseases. Although

across several generations.

**4.3 Immunotherapies**

resource for stem cell and transplantation studies.

**4.3.1 Cancer immunotherapy using heat shock protein**

**4.3.2 Immunotherapy using monoclonal antibodies**

satisfactory for short-term applications, antibody intravenous infusion is not appropriate in many long-term treatments. Fang et al. (2007) describe a recombinant adeno-associated virus (rAAV) gene delivery system that allows regulated long-term expression of native fulllength mAbs *in vivo*. In this study a F2A sequence adjacent to a furin cleavage site (ΔK)RKRR was used to link the antibody heavy and light chain sequences. Notably, the gene expression system included a rapamycin-regulated promoter that can be used to stop mAb production if treatment needs to be terminated. This system potentially offers patients a lifelong mAb therapy that requires only a single administration of an rAAV vector.

Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152, is a coinhibitory molecule that functions to regulate T-cell activation. Antibodies that block the interaction of CTLA-4 with its ligands CD80 (B7-1) and CD86 (B7-2) can enhance immune responses, including anti-tumour immunity (for a brief review, see Chambers et al., 2001). Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a bone marrow growth factor for APCs, which has also been shown to enhance anti-tumour immune responses. Both preclinical animial models and early clinical development indicate synergy between GM-CSF tumour cell vaccination and CTLA-4 blockade (Hurwitz et al., 2000; Hodi et al., 2003; Quezada et al., 2006). To avoid anti-CTLA-4 side effects, tumour cell lines expressing the full-length F2A anti-CTLA-4 mAb in addition to GM-CSF, were administered locally at the immunization site (Simmons et al., 2008). Preliminary results suggest that the delivery of mAbs or proteins locally from immunotherapy cells should prove useful based on promising anti-tumour responses and the reduction of toxicity or adverse immune events associated with systemic exposure.

#### **4.3.3 Cytokines and immunotherapy**

The cytokine Interleukin-12 (IL-12) is a growth and maturation factor acting on both the innate and adaptive arms of the immune system. It is produced primarily by APCs and exerts immunoregulatory effects on natural killer (NK) and T cells (Kobayashi et al., 1989; Wolf et al., 1991). The APC-derived IL-12 consists of two subunits, p40 and p35, which are covalently linked (Kobayashi et al., 1989). The expression of this cytokine has been complicated by the observation that p40 homodimers (in excess of the heterodimer) exhibit antagonistic activity (Trinchieri et al., 2003). To ensure the equal expression of both subunits, biologically active IL-12 protein was produced using F2A as a linker between the p40 and p35 subunits (Collins et al., 1998; Kokuho et al., 1999; Chaplin et al., 1999; de Rose et al., 2000; Premraj et al., 2006). Numerous studies have been done which clearly indicate that plasmid expressed F2A IL-12 can modulate and augment the immune response elicited by DNA vaccination against mycobacterial infections (Triccas et al., 2002; Palendira et al., 2002; Martin et al., 2003). Additionally, it has been reported that IL-23 (but not IL-27) increased protection after *M. tuberculosis* challenge (Wozniak et al., 2006). In this study, the genes encoding p19 and p40 chains of IL-23 and EB13 and p28 chains of IL-27 were cloned on either side of the F2A protein.

Enhanced persistence of adoptively transferred tumour-infiltrating lymphocytes has been demonstrated by the administration of growth cytokines such as IL-2 and IL-15 (for reviews see Westwood & Kershaw, 2010; Ngo et al., 2011). However, systemic toxicity and expansion of unwanted cell subsets, such as regulatory T cells limit the use of these cytokines when administered systemically. Transgenic expression of IL-2 and IL-15 has been shown to increase antigen-specific T cell expansion *in vivo* and enhance antitumour activity without systemic toxicity in preclinical models (Quintarelli et al., 2007). The 3 genes coexpressed in the cytokine encoding vectors (*iCasp-9,CD34,* and *IL-2* or *IL-15*) were linked using F2A. The truncated form of human CD34 was used as a selectable marker of transduced cells and the inclusion of an iCasp-9 "safety-switch" ensured long-term safety of adoptively transferred lymphocytes.

#### **4.4. Gene therapy**

#### **4.4.1** *In vivo* **gene therapy**

Gene therapy can be defined as the introduction of nucleic acids to somatic cells for a therapeutic purpose (Ylä-Herttuala & Alitalo, 2003). Compared to traditional medicine, gene therapy offers unique possibilities to treat the genetic causes of diseases, such as fatal enzyme deficiencies. Mucopolysaccharidosis type 1 (MPS-1; Hurler syndrome) is a congenital deficiency of -L-iduronidase (IDUA), leading to lysosomal storage of glycosaminoglycans. As accumulation and storage continue, tissue and organ damage becomes manifest as loss of function. Patients with MPS-1 present early in life with rapidly progressing disease that usually results in death due to neurological/CNS deterioration and/or cardiovascular/respiratory problems (Neufeld, 1991).

Fig. 5. Schematic of the tricistronic vector construct containing the therapeutic human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes is shown at the top. (A) Whole-organ DsRed2 expression. (B) Cellular DsRed2 expression. (C) Whole-body *in vivo* luciferase imaging. Representative animals of tricistronic plasmidinjected (left), control (*IDUA* injected, middle), and monocistronic luciferase-injected (right) recipients are shown (adapted from Osborn et al., 2005). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

antigen-specific T cell expansion *in vivo* and enhance antitumour activity without systemic toxicity in preclinical models (Quintarelli et al., 2007). The 3 genes coexpressed in the cytokine

form of human CD34 was used as a selectable marker of transduced cells and the inclusion of an iCasp-9 "safety-switch" ensured long-term safety of adoptively transferred lymphocytes.

Gene therapy can be defined as the introduction of nucleic acids to somatic cells for a therapeutic purpose (Ylä-Herttuala & Alitalo, 2003). Compared to traditional medicine, gene therapy offers unique possibilities to treat the genetic causes of diseases, such as fatal enzyme deficiencies. Mucopolysaccharidosis type 1 (MPS-1; Hurler syndrome) is a congenital deficiency of -L-iduronidase (IDUA), leading to lysosomal storage of glycosaminoglycans. As accumulation and storage continue, tissue and organ damage becomes manifest as loss of function. Patients with MPS-1 present early in life with rapidly progressing disease that usually results in death due to neurological/CNS deterioration

Fig. 5. Schematic of the tricistronic vector construct containing the therapeutic human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes is shown at the top. (A) Whole-organ DsRed2 expression. (B) Cellular DsRed2 expression. (C) Whole-body *in vivo* luciferase imaging. Representative animals of tricistronic plasmidinjected (left), control (*IDUA* injected, middle), and monocistronic luciferase-injected (right) recipients are shown (adapted from Osborn et al., 2005). (For interpretation of the references

to colour in this figure, the reader is referred to the web version of this article.)

*CD34,* and *IL-2* or *IL-15*) were linked using F2A. The truncated

encoding vectors (*iCasp-9,*

**4.4.1** *In vivo* **gene therapy**

**4.4. Gene therapy**

and/or cardiovascular/respiratory problems (Neufeld, 1991).

Enzyme may be delivered by enzyme replacement therapy (ERT), haematopoietic cell transplantation (HCT) or by gene therapy vectors (Tolar & Orchard, 2008). P2A and T2A were utilized to construct a tricistronic vector bearing the human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes (*IDUA*-P2A-luciferase-T2A-DsRed). Efficient cleavage was observed and all three proteins were functional *in vitro* and *in vivo*, leading to high-level therapeutic gene expression in NOD/*scid* mice that could be tracked by non-invasive whole-body luciferase imaging and at the cellular level using DsRed2 (Fig.5. Osborn et al., 2005).

#### **4.4.2** *Ex vivo* **gene therapy**

To improve patient safety and increase the gene transfer efficiency, target cells are taken from the patient, gene-engineered and then adoptively transferred into the patient. Redirecting T cell specificity by T cell receptor (TCR) gene transfer is emerging as an attractive strategy to treat patients suffering from malignant and viral diseases. TCR, together with the CD3and signaling subunits, determines the specific CD4+ and CD8+ T cell responses to antigens bound to MHC molecules (Call & Wucherpfennig, 2005; Rudolph et al., 2006). Using the TCR:CD3 complex as a test system, Szymczak and coworkers reported expression of all four proteins that make up CD3 and the two proteins required to make up TCR using just two retroviral vectors (CD3-2A and TCR-F2A) (Szymczak et al., 2004; reviewed in Radcliffe & Mitrophanous, 2004). Following the seminal paper of Szymczak et al in 2004, several groups have reported efficient TCR expression using 2A peptide linkers to combine TCR and - chain genes (Holst et al., 2006a & b; Scholten et al., 2006; Yang et al., 2008; Leisegang et al., 2008; Wargo et al., 2009). An important consideration in redirecting T cells using TCR genes is the tendency of introduced TCR genes to mispair with endogenous TCR - and - chains. In this regard, "murinized" receptors improved HLA-A2/LMP2-TCR expression on the surface of human T cells and downregulated expression of endogenous TCRs (Hart et al., 2008).

The feasibility of TCR gene therapy was recently demonstrated in the first bench to bedside experiments with TCR gene-modified T cells in melanoma patients. Johnson et al., (2009) treated metastatic melanoma patients with autologous T cells genetically modified with retroviral vectors to express high-avidity TCRs recognizing tumour-associated antigens MART-1 (MART-1*TCR-*furinT2A-MART-1*TCR* and gp100 (gp100*TCR*IRESgp100*TCR*). Objective cancer regression was observed in 30% - 19% respectively, of patients who received these high affinity TCRs. However, in the study with TCR targeting MART, some patients also experienced toxicity to normal melanocytes in the skin, eye and ear. Another interesting recent study details the first clinical trial involving the adoptive transfer of engineered lymphocytes with optimal TCR complementary determining regions (CDRs) directed against NY-ESO-1, a cancer-testis antigen frequently expressed in melanoma as well as a wide range of non-melanoma epithelial malignancies (Robbins et al., 2011). In contrast to MART-1 and gp100, which are expressed in normal tissues as well as tumours, NY-ESO-1 expression is limited to neoplastic cells and germ line tissue (Chen et al., 1997). The - and -chains were expressed in retroviral constructs that contained a furin cleavage site followed by a SGSG spacer and the P2A sequence between the two gene products (Robbins et al., 2008). Response rates of 45% and 67% were observed in patients with melanoma and synovial cell sarcoma, respectively, all of whom had progressive disease after extensive prior treatment.

#### **4.5 Induced pluripotent stem cell generation**

Embryonic stem (ES) cells have the ability to differentiate into any cell type of the body and to grow indefinitely while maintaining pluripotency. Remarkably, adult somatic cells can be reprogrammed and returned to the naive state of pluripotency seen in embryonic stem cells by ectopic expression of a defined set of transcription factors: Oct 3/4, Sox2, KLF4 and c-Myc (Takahashi & Yamanaka, 2006; Takahashi et al., 2007; for review see Das & Pal, 2010). The delivery of these "Yamanaka factors" to create induced pluripotent stem (iPS) cells has typically required multiple individual viral vectors, carrying the risk of both insertional mutagenesis and viral reactivation (Takahashi and Yamanaka, 2006; Aoi et al., 2008).

Fig. 6. Schematic diagram of reprogramming cassette. The four reprogramming factors *c-Myc*, *Klf4*, *Oct4* and *Sox2* were fused in-frame *via* 2A sequences and coexpressed as a single ORF and inset: the CAG enhancer/promoter was used to drive the 2A-linked reprogramming cassette and *mOrange* marker, flanked by *loxP* sites (Kaji et al., 2009).

Preliminary work by Okita et al. (2008 & 2010) achieved reprogramming of murine embryonic fibroblasts using repeated transient expressions of two plasmids - one encoding Oct 3/4, Sox2, KLF4 separated by F2A and the other encoding c-Myc. Although the efficiency of iPS cell generation was low, no vector DNA was stably integrated into the iPS cell genome. Sommer et al. (2008) and Carey et al. (2009) reported the derivation of iPS cells from adult skin fibroblasts using polycistronic lentiviral vectors. Sommer's team used a

Embryonic stem (ES) cells have the ability to differentiate into any cell type of the body and to grow indefinitely while maintaining pluripotency. Remarkably, adult somatic cells can be reprogrammed and returned to the naive state of pluripotency seen in embryonic stem cells by ectopic expression of a defined set of transcription factors: Oct 3/4, Sox2, KLF4 and c-Myc (Takahashi & Yamanaka, 2006; Takahashi et al., 2007; for review see Das & Pal, 2010). The delivery of these "Yamanaka factors" to create induced pluripotent stem (iPS) cells has typically required multiple individual viral vectors, carrying the risk of both insertional

mutagenesis and viral reactivation (Takahashi and Yamanaka, 2006; Aoi et al., 2008).

Fig. 6. Schematic diagram of reprogramming cassette. The four reprogramming factors *c-Myc*, *Klf4*, *Oct4* and *Sox2* were fused in-frame *via* 2A sequences and coexpressed as a single

reprogramming cassette and *mOrange* marker, flanked by *loxP* sites (Kaji et al., 2009).

Preliminary work by Okita et al. (2008 & 2010) achieved reprogramming of murine embryonic fibroblasts using repeated transient expressions of two plasmids - one encoding Oct 3/4, Sox2, KLF4 separated by F2A and the other encoding c-Myc. Although the efficiency of iPS cell generation was low, no vector DNA was stably integrated into the iPS cell genome. Sommer et al. (2008) and Carey et al. (2009) reported the derivation of iPS cells from adult skin fibroblasts using polycistronic lentiviral vectors. Sommer's team used a

ORF and inset: the CAG enhancer/promoter was used to drive the 2A-linked

**4.5 Induced pluripotent stem cell generation**

single multicistronic mRNA containing an IRES element separating two fusion cistrons - Oct4 and Sox2 linked *via* F2A and KLF4 and c-Myc linked *via* E2A. In a different way, the Carey group delivered the four factors in a single vector: Oct4, Sox2, KLF4, and c-Myc separated by three different 2A peptides (P2A, T2A and E2A, respectively). Both groups demonstrated reprogramming of fibroblasts to an ES cell-like state, however, in neither case was the polycistronic vector deleted from the genome. Therefore, attempts were made to minimize genome integration by removal of the inserted genes after the reprogramming process was switched-on (Chang et al., 2009; Kaji et al., 2009; Woltjen et al., 2009; Yusa et al., 2009) and more recently by using mRNA/miRNA of the four factors rather than DNA vectors (Warren et al., 2010; Yakubov et al., 2010; Miyoshi et al., 2011). The efficient reprogramming of murine and human embryonic fibroblasts and the traceless removal of factors joined with viral 2A sequences by using the Cre/LoxP or *piggyBac* transposon/transposase systems mark important advances towards achieving clinically acceptable methods of deriving iPS cells (see Fig.6).

#### **5. Looking ahead**

Exciting work of many laboratories in the last few years has clearly established the importance of 2A for co-expression technology. Our increasing knowledge about the cleavage mechanism indicates 2A is not just a novel method of controlling protein biogenesis, but that a crucial aspect of its function is to act as a translational "sensor". Protein synthesis in eukaryotes consumes a high proportion of cellular energy, most of which is used in elongation. During times of energy and/or nutrient deprivation, 2A could act to terminate translation in a stop codon-independent manner – devoting the remainder of the cell's resources into translating only that portion of the ORF upstream of 2A. We envisage that 2A-mediated cleavage could find extra utility in the biomedical and biotechnology fields as a reporter for translational stress.

#### **6. Acknowledgements**

The long term support of the Wellcome Trust and the Biotechnology and Biological Sciences Research Council is gratefully acknowledged. The University of St Andrews is a charity registered in Scotland no. SCO13532

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