Gene Knockdown Therapy

a. Antisense Drugs. Antisense technology is a novel gene delivery method that is increasingly applied to knock down the expression of a specific target gene for therapeutic purposes or to study the function of that gene. The fundamental principle of the antisense approach is to silence a gene using a short synthetic DNA or RNA sequence that is homologous to that contained within the target gene. Antisense oligodeoxynucleotides (ODNs) are synthesized in the opposite direction of the known complementary DNA sequence and are designed to hybridize specifically with their target sequences to interrupt the production of the corresponding protein. Almost all human diseases are associated with a dysfunctional protein. While most conventional drugs are designed to inhibit the disease-causing activity of a dysfunctional protein, antisense molecules are designed to inhibit the production of the protein. In principle, gene silencing may be accomplished at the genetic level by inhibiting biological events, such as transcription, translation, or gene splicing (180183). During the inhibition of transcription events, ODNs bind to double-

stranded DNA to induce the formation of a short triple-helical structure. This structure is mediated by Hoogsteen hydrogen bonds and sterically hinders the transcription of a specific mRNA. In addition, translation of RNA species can be interrupted by binding of ODNs to tRNA or pre-RNA to prevent their transport from the nucleus or by directly interacting with target mRNA molecule after transcription. In cases where inhibition occurs after the transcript is matured, antisense binding to RNA is intended to block ribosomal assembly or ribosomal sliding along the mRNA during translation of the protein. ODNs can also be of therapeutic value if they are designed to target the intron-exon junctions of premature RNA. In this regard, they prevent splicing events that are essential for maturation of the RNA transcript. Three regions of the RNA that are considered the best targets when designing ODNs are the 5 ' Cap region, the AUG translational initiation codon, and the 3 ' untranslated region.

The concept of disabling the function of a mRNA by hybridization of antisense reagents is a simple one, but, like other gene-based therapies, the technology has encountered difficulties in the past. The technical problems experienced in the early pioneering stages of antisense technology are only now being elucidated and are the focus of active study. From these analyses, several features are apparent in the design of effective antisense molecules: determining the length of sequence with the greatest activity and specificity; cellular uptake; specific targeting of the ODN; antisense stability; and toxicity. Other factors that have influenced the effectiveness of antisense molecules are frequency of protein turnover, the intracellular environment of the cell, and the extent of longevity of ODNs after administration.

Gene knockdown practices are still under development and will require significant modifications before being clinically acceptable as a therapeutic modality. Introducing variations in antisense chemistry by subtle changes in the phosphate or sugar moieties of the nucleic acid backbone is one method that shows success in minimizing nuclease degradation of the molecules. Replacing a nonbridging oxygen with a sulfur atom in the phos-phodiester bond between nucleotides on the phosphate backbone generates a phosphorothioate linkage, which is reported to be one of the most successful modification of antisense oligonucleotides to date (184,195). Phosphorothioate compounds have shown efficacy in delivery and are less vulnerable to intracellular nuclease degradation. A disadvantage of their use, however, is that the constructs are chiral and form a racemix mixture of ODN species that exhibit both desirable and undesirable properties in vivo (186). Some ODNs are reported to be toxic, while others show nonspecific affinity for proteins (1987). The technical progress in chemical modification of antisense has recently shifted from the first-generation phosphodiester oligonucleotides, which are still nuclease sensitive, to the more nuclease-resistant chimeric compounds that contain methoxyethyl modifications at the end of the ODNs. The development of oligonucleotide conjugates with cell-penetrating and nuclear-targeting peptides and colloidal antisense carriers that protect against degradation is emerging rapidly and will significantly improve cellular uptake, stability, subcellular trafficking, and increased in vivo activity (188-192). Over 200 patents disclose antisense sequences with therapeutic utility in the treatment of human diseases. It is a powerful tool with exceptional clinical value and is being exploited to identify gene function and validate new drug targets.

Formivirsen (ISIS 2922) is the first antisense oligonucleotide drug approved for the treatment of cytomegalovirus (CMV)-induced retinitis. The 21-phosphorothioate oligonucleotide inhibits viral replication in the human eye by binding to complementary sequences of early mRNA CMV viral transcripts. In preliminary clinical trials, the progression of CMV retinitis in AIDS patients is significantly delayed after intravitreal administration of formivirsen. Drug-clearance studies show that formivirsen exhibits first-order kinetics with a half-life of 62 hours in rabbits and 78 hours in monkey. A mild and transient inflammatory response and increase in intraocular pressure are observed after treatment with formivirsen. These appear to be resolved spontaneously or reversed with topical steroid treatment (193-196).

Diseases characterized by retinal neovascularization are among the principal candidates for antisense treatment. The use of antisense oligonu-cleotide against vascular endothelial growth factor (VEGF) has shown promising results for the treatment of proliferative retinopathy. After intraocular administration in a murine model of retinal neovascularization, phosphorothioate antisense molecules reduced VEGF protein synthesis and the growth of new blood vessels in a dosage-dependent manner. The study shows the therapeutic potential of ODNs in ischemia-induced proliferative retinopathies (197). Proliferative vitreoretinopathy (PVR) is an ocular disorder often associated with proliferating RPE cells. Antisense knockdown of c-myc, a protein active in the mitogenic pathway, inhibits the proliferation of human retinal pigment epithelial cells, suggesting that c-myc ODNs may be an exciting perspective in the treatment of PVR (198).

Retinal ganglion cell death is associated with increased expression of the Bax protein after transection of the optic nerve. A phosphorothioate Bax antisense oligonucleotide was reported to show therapeutic utility in preserving ganglion cell following axotomy. Bax expression was reduced and the number of surviving neurons increased after treatment with Bax ODNs. This represents a novel approach for neurodegeneration due to optic nerve injury (199). The use of ODNs to silence the expression of another retinal gene GLAST, a glial glutamate transporter, showed sig nificant changes in normal retinal transmission and indicates the importance of GLAST in maintaining retinal function (200). Similarly, an antisense compound generated against the trkB receptor mRNA for brain-derived neurotrophic factor (BDNF) alters the neurochemical phenotype of retinal neurons (201). BDNF and its receptor are important to survival and differentiation of the retina and are potentially useful targets in retinal degenerative diseases. Antisense targeting of fibronectin transcripts was also shown to reduce the expression of fibronectin in retinal vascular cells (202). The use of antisense oligonucleotides in these studies reflects the significance of the technology in understanding the function and regulation of a specific protein and the potential therapeutic benefits for antisense-based ocular therapies.

b. Ribozymes. Ribozymes are naturally occurring catalytic RNA and a new class of genetic tools used to inhibit gene expression. Designer ribozymes are chemically designed to recognize and bind specific RNA through complementary base-pair hybridization. Their value in human therapeutics is dependent on their ability to distinguish between mutant and wild-type RNA species and to act as molecular scissors to digest or edit the target RNA in a way that will prevent translation of the corresponding protein (203-205). There are developed as an alternate approach to antisense drugs. Analysis of the physical, biochemical, and biological properties of naturally occurring ribozymes has allowed researchers to classify them according to their various catalytic functions:

1. Hammerhead ribozymes: These are approximately 30 nucleotides long and the smallest ribozymes identified. They are found in many viral DNA and are capable of site-specific cleavage of a phosphodiester bond. Hammerhead ribozymes have been extensively studied, and many have been synthesized against RNA targets. In recent years they have emerged as a potentially effective therapeutic measure in models of retinitis pigmentosa. In areas of the brain, hammerhead ribozymes have been directed against the amyloid peptide precursor (B-APP), which is associated with the pathogenesis of Alzheimer's disease. Others, such as angiozyme, have been synthesized against angiogenic processes involved in the progression of tumor metastasis.

2. Group 1 and Group 11 intron ribozymes: These species can self-splice, digest, and ligate phosphodiester bonds. They are found in lower eukaryotes and some bacteria. Group 1 intron ribozymes mediate trans-splicing of RNA targets and is considered a useful genetic tool in repairing mutations in defective genes.

3. Ribonuclease P: Cleaves phosphodiester bonds of tRNA precursor molecules.

The catalytic activity of these molecules make them particularly interesting in the treatment of dominantly inherited diseases. In autosomal dominant retinitis pigmentosa (ADRP), a substitution of histidine for proline occurs at codon 23 in the rhodopsin gene. This mutation is referred to as P23H and is responsible for the synthesis of a mutant gene product that results in the death of photoreceptor cells (206). Because the field is relatively new, only a few studies have been carried out in the retina to test the therapeutic effect of ribozymes in ocular diseases. One research team has now shown that in vivo expression and activity of hairpin and hammerhead ribozymes can be achieved in a transgenic rat model of ADRP. Efficient transduction and stable expression of the ribozymes were accomplished using an adeno-asso-ciated virus that contained a rod opsin promoter. The results suggested that the expressed ribozymes discriminated between wild-type and mutant rho-dopsin RNA and specifically destroyed the P23H mutant specie. As a result, translation of the P23H protein was inhibited and progression of photore-ceptor degeneration in ADRP model was significantly slowed down (206212). Combining the advantages of current gene delivery strategies with catalytic ribozymes has broad therapeutic implications for dominantly expressed retinal diseases where the disease is already in progression.

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