Department of Physics, Chemistry and Biology (IFM)

Biosynthetic corneal constructs and encapsulation

The cornea is an avascular, transparent and immuneprevileged tissue that consists of three layers: an outer stratified epithelial cell layer, a middle stromal cell layer, and an inner endothelial cell layer (Fagerholm et al, 2010;   Li et al, 2003;   Liu et al, 2006) .Corneal blindness is defined by the World Health Organization as a visual acuity of 3/60 or less. There are a range of diseases that can lead to corneal blindness, such as keratoconus, autoimmune disease, trauma, burns, recurrent infections and graft rejection. Transplantation with donor corneal tissues is the treatment for many of the conditions. However there is a severe shortage of donor corneas worldwide leaving over 10 million patients on the waiting list for a transplant (Whitcher et al, 2001) . In addition, conditions such as autoimmune disease, chemical burns and preiously rejected grafts do not respond well to allograft transplantation.

Griffith et al,. developed a biosynthetic corneafrom1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linked recombinant human collagen (Fagerholm et al. 2010), which was tested in 10 patients, and shown to promote regeneration of the human cornea. This is therefore a possible future alternative to human tissues for transplantation.

However, this biosynthetic cornea was only tested in keratoconus and scarred corneas, and not corneas with pre-existing infections such as Herpes Simplex Virus-1 (HSV-1) infections that often remain latent in the nerves of the cornea and can be reactivated during the transplantation to cause tissue destruction in the new allograft. To deal with HSV-1 infected corneas, we therefore need to ensure that implants that are developed are equipped to prevent viral reactivation.

Herpes simplex virus types and its diseases 

Herpesviridae, a member of the alpha herpes virus family, consists of over 80 DNA viruses. There are only 8 members of herpes virus that are known to infect humans, of which herpes simplex virus type 1 and 2 (HSV-1 andHSV-2) affect 60 to 90% of the population. These viruses are also called human herpes viruses (HHV) (Polcicova et al, 2005;Wuest et al, 2011;   Yoneda et al, 2011) . HSV-1 and HSV-2 are known to be neurotrophic and neuroinvasive viruses which makes them capable of inhabiting a neuron or ganglion and remain latent until an external or internal stimuli causes them to reactivate (Di Giovine et al, 2011;   Koelle & Wald, 2000;   Polcicova et al, 2005;   Remeijer et al, 2004;Szpara et al, 2011;   Vastag et al, 2011;   Yoneda et al, 2011) .HSV-1 is commonly associated with cold sores or fever blisters whereas HSV-2 is often associated with genital infections (Szpara et al, 2011) .HSV reactivation can be stimulated by physical or emotional stress, fever, exposure to UV light, immune suppression, and corneal surgeries such as LASIK (Koelle & Wald, 2000;   Polcicova et al, 2005;   Remeijer et al, 2004) .HSV-1 infection can cause different types of ocular diseases; some of these infections can be an extreme visual threat to the infected host, such as herpetic stromal keratitis(HSK) which can lead to corneal blindness (Shah et al, 2010) .HSK is characterized by destruction of the stromal layer of the cornea which leads to neovascularization and opacification of the normally transparent cornea, thereby resulting in diminished vision (Polcicova et al, 2005;Rajasagi et al, 2011;   Sharma et al, 2011) .HSV-1 infection in the cornea leads to chronic inflammation, which is mediated by cytokines and chemokines, secreted by T-lymphocytes. As the cornea is an immunoprivileged organ, the mechanism through which the cytokines are recruited to site of infection is still unknown (Chang et al, 2000;   Deshpande et al, 2001;   Newell et al, 1989) .

HSV structure and its replication 

HSV-1 is alarge and complex DNA virus. The HSV-1 virion has a well defined structure composed of four components: (1) Core – containing double-stranded viral DNA with approximately 152 kbp, (2) Capsid – consists of 162 capsomers arranged in an icosadeltahedron, (3) Tegument – various thickness of protein layers and enzymes including virion host shut-off (VHS) proteins which surrounds the capsid, and (4) Envelope – the whole structure is enclosed by a viral envelope consisting of a lipid bilayer with glycoproteins. There are ten known HSV-1 glycoproteins that act as attachment proteins, fusion proteins, structural support and immune escape proteins (Kaye & Choudhary, 2006;   Whitley et al, 1998) . These proteins are required for HSV-1 to enter into the host cell and induce infection. The virus adheres to the receptors on the cell surface, after which its envelope fuses with the plasma membrane. Subsequently, the de-enveloped capsid is transported to the nuclear pores and releases its DNA into the nucleus (Kaye & Choudhary, 2006;   Whitley et al, 1998) .

After entering the nucleus, the virion host shut-off (VHS) protein stops the synthesis of host macromolecules thus stimulating the transcription and viral replication by the help of host polymerases. The transcription, replication of viral DNA and assembly of new capsids take place in the nucleus.There are three major types of genes or proteins, namely α, β and γ that are expressed after infection.These genes are involved in the process of regulation, transcription, replication and production of viral glycoproteins. The enveloped capsids containing newly synthesized DNA attaches to the nuclear surface of the inner nuclear membrane and releases DNA into the space between the inner and outer nuclear membrane. The enveloped viruses are organized to diffuse from cell to cell by local spread. The viral particles can therefore migrate along the sensory nerves to exhibit latent or active infections at other sites (Kaye & Choudhary, 2006;   Remeijer et al, 2004;   Whitley et al, 1998) .

Anti-viral compounds  

HSV-1 mediated corneal blindness is a big problem worldwide. To treat this infection there are many anti-viral compounds approved for medical use. The antiviral activity exerted by these compounds affects viruses of the herpes family, including HSV 1 and 2. These compounds selectively block the viral replication by binding to the DNA helix and thus disturbing the processes of replication.Currently, there are many anti-viral agents available for example Fosccarnet, idoxuridine, acyclovir and trifluridine (Wilhelmus, 2010) . 

Initially, pyrimidine and purine analogues were developed as anti-infective compounds against HSV.  Later, some natural compounds derived from plants were used for the treatment of several viral diseases. Consequently, both were replaced by synthetic nucleoside and non nucleoside analogues such as acyclovir (ACV), biruvidin (BVDU), trifluridine(TFT) etc. These analogues are less toxic when compared to other anti-viral agents because they are prepared by thymidine kinase, viral encoded enzyme to slow down viral DNA polymerase activity (Larsson & Öberg, 1981;   Wilhelmus, 2010;Yoneda et al, 2011) . 

In general, compoundswhich have several condensed indole rings possess antiviral activity and they are structurally related to ellipticine and quinacrines. Earlier studies showed that ellipticine, quinacrines and its derivatives have antiviral and antitumor properties and the ability to intercalate into DNA (Harmenberg et al, 1988;   Sehlstedt et al, 1998) .

Some anti viral compounds are not sufficiently effective, have restricted action due to host toxicity and may results in viral gene mutations causing resistance (Larsson & Öberg, 1981;   Wilhelmus, 2010;Yoneda et al, 2011) . Acyclovir was the first anti-viral drug that was proven to inhibit viral replication with relatively low level of toxicity to the cells.  However, drug resistant HSV-1 has now been documented in HSV patients (Marques & Straus, 2000;   Weber & Cinatl, 1996) . The mechanism of resistance might be due to mutation causing insufficiency of viral thymidine kinase (TK) enzyme and/or viral polymeras as acyclovir is a nucleoside analogue which retards viral DNA elongation.

In order to avoid these narrow spectrums of antiviral activity, there is a necessity for developing new antiviral drugs that do not rely on blocking TK activity. The antiviral compounds evaluated in this study are derivatives from the B220 (2,3-dimethyl(dimethyl aminoethyl)5H-indolo-(2,3-b)quinoxaline ) compound that previously has been shown to be a potent inhibitors of viral replication (Wilhelmsson et al, 2008) . The replication of HSV-1, viral DNA synthesis and formation of both early and late viral proteins were inhibited by this compound and its derivatives, e.g. hydroxylated B220 (9-OH-B220) (Harmenberg et al, 1988;   Sehlstedt et al, 1998) .