Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1) can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.
Gene therapy is the experimental use of genetic manipulation techniques to correct errors associated with genetic diseases or to modify undesirable Deoxyribonucleic acid (DNA) sequences. The ever extending list of genetic diseases opens the door wide to gene therapy as a new hope for targeting the aetiology rather than the symptoms of these diseases. Plenty of disciplines of gene therapy are currently discussed in the literature. However, there is a general agreement on few main issues to be thoroughly addressed before commencing a clinical trial for a novel gene therapy. Those include the precise diagnosis of the addressed genetic error, the relation between the causative gene defect and the resultant disease, the specific targeted tissue in the body, the dosage form design, and the choice of route of administration. Gene therapy approaches to corneal pathological disorders are being studied extensively to provide much needed progress against specific corneal malfunctions. Unlike protein based therapy, gene therapy has more research-attractive benefits being cheaper, better controlled, and more efficient in many occasions.
Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen that causes life-long recurring disease. Two HSV serotypes exist, HSV-1 and HSV-2, with distinct tropism reported for each. The cold sore virus (HSV-1) is a leading cause of corneal blindness [
As of January 2012, the online record gene therapy clinical trials worldwide provided by the Journal of Gene Medicine (
HSV-1 belongs to the human herpes virus (HHV) family. The HSV-1 virion is 120–300 nm in size. The genetic material of HSV-1 comprises 152,000 base pairs (encoding more than 80 genes) arranged as a double-stranded DNA, which circularizes upon infection [
HSV-1 model structure and genome arrangement. (a) The icosahedral, DNA-containing capsid is asymmetrically located within the virion and surrounded by an amorphous protein layer called the tegument, and a membrane envelope heterogeneously studded with morphologically distinct spikes formed by 12 different glycoprotein species. (b) The HSV-1 genome arrangement showing repeats surrounding UL designated ab and b′a′, and those surrounding US designated a′c′ and ca. There are two different origins of replication, oriL in the long segment and oriS in the short segment. Abbreviations: UL: long unique sequence; US: short unique sequence; TRL: terminal repeats of long segment; TRS: terminal repeats of short segment; IRL: internal repeat of the long segment; IRS: internal repeat of short segment.
During the latency stage of the HSV lifespan, the expression of latency-associated transcripts (LATs) is switched on, while general gene expression is restricted. Exit of the dormancy state can be triggered by decreased immunity due to infection, stress, ultra violet (UV) radiation or fever. The conjunctiva is believed to be the first affected organ by active HSV-1 infection [
Regarding the fact that asymptomatic HSV-1 infection is widespread, the diagnosis of HSV-1 through clinical symptoms is questionable. Laboratory methods performed by a specialized virologist is highly recommended [
Treatment of HSV-1 cases typically combines medications such as inflammation, immune and neovascularisation suppressing agents together with an anti-viral drug. Corticosteroids are used to improve clinical signs due its anti-inflammatory and antiangiogenesis effects. Several antiviral drugs have shown efficacy in the treatment of ocular HSV-1 keratitis, including acyclovir, valacyclovir, cidofovir, trifluorothymidine, and ganciclovir [
Understanding the mechanisms by which HSV-1 can survive or overcome the host immunity is a gateway for the development of novel antiviral therapies. Primarily, complex anti-viral defence mechanisms based on innate and adaptive immunity activates immune recruitment mechanisms. An immune cascade, orchestrated mainly by T-cells, is responsible for the pathogenesis of HSV-1. Other antiviral resistance molecules, grouped under “intrinsic antiviral immunity”, have recently emerged as a promising class in virus-battling research field [
Three approaches of gene transfer have been considered. The first one is systemic administration, even if the wide spread distribution in the host body is a major concern. Moreover, the gene delivery to the trigeminal ganglion where the HSV-1 latency requires high specificity and crossing the blood brain barrier to nerves. Second is the local application of naked therapeutic gene or a loaded vector. The third approach is to treat the cornea
The vectors must be chosen and modified to safely and efficiently escort DNA from outside the cell to the nucleus and to overcome several physical barriers that are obstacles to internalization, escape from endocytotic vesicles, movement through the cytoplasm, and transport into the nucleus. Vehicles for ocular gene therapy have been described in a broader prospect in number of reviews, therefore is not the focus of this paper. Nonetheless, here we attempt to fill the gaps, concentrate on the latest advances, and set up future directions for each vector type that can be used for corneal gene therapy of corneal herpetic keratitis.
Most clinical trials for ocular gene therapy utilized viral vectors for gene delivery [
The current leading choice for corneal gene therapy is the adeno-associated virus (AAV). The expression pattern of this virus shows a delayed expression of the transgene; therefore, the therapeutic activity can be sustained for many years [
Lentivirus and retrovirus belong to the group
The transduction efficiency of AAV can be improved by several methods, for example, creating a pocket with 110
Retrovirus (and lentivirus) genome is single stranded RNA, encapsulated in a lipid envelope. The name retro comes from its property of retro-transcription in linear double strand DNA to integrate into the host genome. Retroviral vectors recommended for transducing the cornea are based on different lentiviruses like HIV, equine infectious anaemia virus (EIAV) or feline immunodeficiency virus (FIV). The ability of retrovirus to transduce human corneas was confirmed in several report [
Retroviral vectors are well known to integrate their genome into the host to achieve stable transgene expression. This property is useful when treating chronic HSV-1 infection because of the long lasting gene expression benefit. Genes can be delivered to the endothelium by injection into the anterior chamber of the eye. Most retroviral vectors are genetically modified to isolate the
Neovascularisation resulting from HSV-1 immuno-stimulation causes severe vision opacity. An early study showed that the transfer of naked complementary DNA (cDNA) encoding the vascular endothelial growth factor (VEGF) receptor antagonist to the eye was shown to block the formation of blood vessels [
Intraocular injection of DNA plasmid encoding Interleukin 18 (IL-18) reduced angiogenesis and immunoinflammatory lesions resulting from HSV-1 infection of mice [
Nanoparticles are used in many drug delivery systems. Hyaluronic acid and chitosan nanoparticles can be loaded with genetic medicines, RNAi or DNA plasmids and used for corneal gene therapy. Albumin nanoparticles were able to reduce neovascularisation by 40% after 5 weeks, with no significant toxicity [
Electroporation method employs high field strength, square-wave electric pulses to allow the penetration of therapeutics molecules. Genetic material or therapeutic molecules are applied to the surface of the cornea, and then the electric pulse will assist the diffusion process. While this method does not involve any biochemical agents and is therefore considered a safer choice, electroporation can cause severe cell damage and induce immunogenic reactions [
Applications of the femtosecond laser are becoming more accepted in corneal refractive surgery and transplantation, due to high precision and safety, compared to conventional laser. The femtosecond laser can be used to create a pocket where in the corneal surface to assist the delivery of therapeutic agents. It was used to deliver the vector to the stroma [
Antibody targeting can be a useful addition to the liposome gene transfer techniques. The use of antibodies with certain cell-specificities can be useful to target a specific corneal layer. Liposomes containing plasmid DNA and coated with antibodies will form an immuno-specific vector to target specific receptors of a given cell type, for example, targeting the endothelium to improve allograft survival [
Gene gun method is also known as particle bombardment, microprojectile gene transferor gene gun. Ballistic transfer with the gene gun can be used to transfer cDNA coated gold particles to the epithelium [
Dendrimers are macromolecules characterised by extensively branched three dimensional structure that can accommodate a genetic material, hence used for drug delivery. Activated polyamidoamine dendrimers carrying a DNA plasmid transduced up to 10% of corneal endothelium after direct application to the cornea [
A body of work in gene therapy for corneal disorders has established the potential of finding new anti-HSV-1 gene therapy. Drug delivery to the cornea by gene therapy is preferably applied locally to avoid systemic complications, or performed
Most of the gene therapy approaches to combat HSV-1 were directed to the inflammation process. In 20% of herpetic keratitis infections, when the latent virus is reactivated it results in chronic ocular immune response where specific lymphocytes are produced, mainly CD4+ T producing T-helper 1 (Th1) cytokines [
Chronologic order | Vector | Gene | Host | Rout of administration | Results and reference |
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1990 | Vaccina virus | gD | Rabbits | Intradermal injection | No effect on herpes simplex keratitis during 16 days postinfection [ |
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1997 | Plasmid DNA | IL-10, IL-2, GM-CSF | Mice | Topical or intramuscular | Topical: IL-10, only, reduced lesion severity. Intramuscular: no effect [ |
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1998 | Plasmid DNA | IL-2, IL-4, IL-10, IFN- |
Mice | Topical | IL-4 or IL-10: reduced lesion severity 25 days post-infection; |
Plasmid DNA | IL-2, IL-4, IL-10 | Mice | Topical, nasal, or intramuscular | Topical administration of IL-4 or IL-10 lowered lesion severity 21 days after infection [ | |
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1999 | Plasmid DNA | IFN- |
Mice | Topical | Increased survival if treated 1 day pre-infection [ |
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2000 | Plasmid DNA | gB1 | Rabbits | Subconjunctival or intramuscular injection | Complete survival and reduction of lesions following intramuscular administration only [ |
Plasmid DNA | gD, gD-IL-2 | Mice | Subconjunctival injection | Good survival for grafts and prevention of stromal, but not epithelial, keratitis [ | |
Plasmid DNA | IFN- |
Mice | Topical, nasal, or vaginal administration | Topical application improved the survival when treated 1 day before infection [ | |
Plasmid DNA | IFN- |
Mice | Topical | Increased survival only when applied 1 day before infection [ | |
Plasmid DNA | IFN- |
Mice | Topical | Increased survival only when applied 12 hours before infection [ | |
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2001 | Plasmid DNA | IFN- |
Mice | Topical | Increased survival [ |
Plasmid DNA | gB | Mice | Mucosal | Increased INF- | |
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2002 | Plasmid DNA | gD-IL-2 | Mice | Subconjunctival injection | Complete prevention of stromal, but not epithelial, keratitis 10 days after infection [ |
Plasmid DNA | gD-IL-2 | Mice | Topical conjunctival | Complete prevention of stromal, but not epithelial, keratitis [ | |
Plasmid DNA | IL-12, IP-10 | Mice | Topical | Suppression of lesions using both transgenes. IP-10, but not IL-12, suppressed lesions [ | |
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2003 | Antisense oligonucleotides | TNF- |
Mice | Subepithelial injection | Reduction of herpes simplex keratitis signs 14 days after infection [ |
HSV-1 | IL-2, IL-4, IFN- |
Mice | Intraperitoneal injection | All transgenes: complete survival and prevention of corneal scarring 28 days after infection [ | |
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2004 | Plasmid DNA | gB, gC, gD, gE and gI | Mice | Intramuscular injection | Complete survival and prevention of corneal scarring 28 days after infection [ |
siRNA duplexes with or without TargeTran | VEGF | Mice | Subconjunctival or intravenous injection | Subconjunctival or systemic administrations suppressed lesions 10 days after infection [ | |
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2005 | HSV-1 | IL-12p35, IL-12p40 | Mice | Intraperitoneal injection | Both transgenes: complete survival and prevention of corneal scarring 28 days after infection [ |
Plasmid DNA | IL-18 | Mice | Topical | Suppression of lesions 12–21 days post-infection [ | |
Plasmid DNA encoding shRNA | Matrix metalloproteinase-9 | Mice | Intrastromal injection | Suppression of lesions during 21 days after infection [ | |
2008 | siRNA | TNF- |
Mice | Intraperitoneally | Inhibition of TNF- |
Plasmid DNA | IL-10 | Mice | Topical | Modulate the inflammation severity [ | |
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2009 | Plasmid DNA | VEGFR2 VEGFR3 | Mice | Subconjunctival injection | Control vascularisation after HSV-1 infection [ |
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2010 | Nanoparticles | IL-21 and gD vaccine | Mice | Ocular mucosal administration | Inhibits ocular HSV-1 [ |
DNA plasmid | B and T lymphocyte attenuator | Mice | Intraperitoneal injection | Decreased CD4+ T mediated cell response [ |
Abbreviations—gD: HSV-1 glycoprotein D; GM-CSF: granulocyte-macrophage colony-stimulating factor; INF: interferon; IL: interleukin; IP-10: IFN-inducible protein 10; shRNA: short hairpin RNA; siRNA: small interfering RNA; TNF: tumour necrosis factor; VEGF: vascular endothelial growth factor. Table updated from [
DNA damage response is a mechanism by which cells can correct damage or eliminate severely damaged cells by activating programmed cell death mechanisms. DNA damage mechanisms are involved in processes such as excision of the damaged area, cell cycle arrest to prevent the pass on of mutated sequences or the transcriptional level control. However, severe injuries can cause the cell to undergo apoptosis. The DNA repair mechanisms include direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and cross-link repair. Detailed description of these mechanisms is discussed elsewhere in more detail [
Targeting the HSV-1 genome for degradation, using several techniques have been reported, including the use of ribozymes [
Successful targeting of HSV-1 genome by gene therapy to date. Abbreviations: DNAzymes: deoxyribozymes; siRNA: small interfering RNA; HSV-1: herpes simplex virus serotype 1.
Ribozymes are RNA molecules with intrinsic enzymatic activity to promote a variety of reactions without the aid of protein cofactor, usually involving cleaving or splicing of RNA molecules, therefore, can be used for gene therapy [
Antisense oligodeoxynucleotides are short synthetic DNA consisting of 15 to 20 nucleotides. They inhibit protein biosynthesis by specifically targeting the complementary stretches of RNA. In principle, they are able to interfere with each step of nucleic acid metabolism, preferentially to block translation [
As antisense oligonucleotides, morpholinos function by translational arrest. Phosphorodiamidate morpholino oligomers (PMOs) are a subclass of antisense oligonucleotides modified to include a phosphorodiamidate linkage and morpholine ring to demonstrate limited off-target effects, favourable base stacking, high duplex stability, high solubility, cell permeability, and no hybridization complexities [
The ability of transfected synthetic siRNAs to suppress the expression of specific transcripts is a useful technique to probe gene function in mammalian cells. Plasmids encoding small hairpin RNAs are extensively used in gene therapy. Consequently, siRNA was used to target viruses in several studies [
Oligonucleotide sequences with the capacity to recognize specific target molecules with high affinity and specificity, referred to as “aptamers”, are beginning to emerge as a class of molecules that compete with antibodies in both therapeutic and diagnostic applications. Recent studies have shown
Targeting endonucleases to HSV-1 genome for excising is an emerging new concept for antiviral gene therapy. Homing endonucleases are a group of restriction enzymes encoded by introns and inteins. Their recognition sites are rare, however, custom made endonucleases, namely meganucleases, can be made targeting specific viral sequences for gene therapy [
Several viral components have been proposed as promising targets for antiviral drug discovery by targeting them in a highly selective and effective manner [
The potential in the treatment of many genetic diseases and viral infections is progressively elevating the attention and awareness towards this new era of highly specialized treatment. The fact that gene therapy is now being clinically evaluated, promotes the massive research that is conducted currently in animal models. It is well established that therapeutic genes or molecules can be transferred to the cornea by direct application of naked DNA, electric pulse, ballistic transfer with a gene gun, viral and non-viral vectors, or several creative combinations of these approaches. The choice of the appropriate vector for targeting HSV-1 is essential for successful gene therapy. The expression profile, bioavailability, biodegradability, and specificity of the vector are the main variables currently evaluated by several research groups to achieve the ideal combination. Successful gene therapy requires the choice of the right vector for each particular disease. Choices are made based on the ability of the targeted cells to uptake the vector, expression of the transgene in a cell-specific manner, and maintenance of expression for the period of time needed to tackle the disease. For chronic cases not requiring a surgery, local application to the cornea is usually preferred. However, pretreatment of corneas