| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
| June 2002 | Inside IOVS | Volume 43/6 |
| Burgeoning Molecular Biology and Gene Therapy in the Eye |
A Corneal cDNA Library
Sakai et al. (p. 1749) constructed a 3’-directed complementary DNA library of human corneal endothelium based on analysis of 2663 clones. The abundant transcript genes consist of prostaglandin D2 synthase lactate dehydrogenase-A, a novel gene without a known function, and matrix Gla protein. The authors report a full-length sequence of the novel gene showing the similarity to genes obtained in the ovary and the testis. This complementary DNA library of human corneal endothelium could provide new insights into the physiology of corneal endothelium.
Gene Expression during Corneal Wound Healing
Corneal wound healing is a complex process that is poorly understood at the molecular level. Varela et al. (p. 1772) used microarray technology to characterize alterations in expression of 1176 rat genes following excimer laser injury compared to normal corneas. Expression of 42 genes increased ≥3-fold while 28 genes decreased expression ≤3-fold on day 3, and on day 7 expression of 20 genes was elevated and 7 genes decreased expression. Hierarchical clustering of 588 genes that changed expression produced 10 clusters, with over-representation of functionally related genes in 7 of the clusters. Microarray analysis identified new genes that participate in corneal wound healing.
CYP1B1, Glaucoma, and Cancer
Stoilov et al. (p. 1820) report that CYP1B1 mutations were observed in half of the Brazilians with primary congenital glaucoma (PCG). Such mutations were twice as frequent in individuals of European descent as compared to individuals of African descent. The majority of CYP1B1 mutations were associated with the “high activity” V432 allele, which is emerging as a putative susceptibility factor in several cancers. Because CYP1B1 mutations causing PCG will effectively eliminate the V432 allele, the authors speculate that this may provide a biological advantage to heterozygous carriers of CYP1B1 mutations by reducing their susceptibility to various cancers.
FOXC1 in Glaucoma
FOXC1 is a member of the forkhead family of genes, which have important roles in tissue development. In the eye, FOXC1 mutations result in a spectrum of anterior segment developmental defects that are associated with glaucoma. As reported in Lehmann et al. (p. 1843), the identification of several families with such phenotypes, in which duplications and deletions encompassing FOXC1 respectively increase or decrease the number of copies of FOXC1, demonstrates that altered gene dosage can cause glaucoma.
Gene Expression in HSV-1 Latent Ganglia
Higaki et al. (p. 1862) used a high density gene array for 1185 mouse genes to compare gene expression in trigeminal ganglia from immunosuppressed and untreated HSV-1 latent mice. Expression was significantly increased for 10 genes and significantly decreased for 8 genes. Semiquantitative RT-PCR for selected upregulated and downregulated genes confirmed the array results. Increased expression of genes included prostaglandin E2 receptor EP4 subtype (PTGER4), insulin promoter factor 1 (IPF1), glutathione S-transferase mu 2, cyclin D2, peripherin, plasma glutathione peroxidase, methyl CpG-binding protein 2, retinal S-antigen, and ErbB2 proto-oncogene. These are suggested to be of special importance for HSV reactivation induced by immunosuppression.
Differential Display in Cataract
The Emory mouse is a well-studied model for age-onset cataract; however, the cause of cataract in the Emory mouse is not precisely defined. Sheets et al. (p. 1870) used differential display to identify candidate genes whose expression levels are altered between pre- and post-cataract Emory mouse lenses. The authors confirmed decreased levels of crystallins between pre- and post-cataract Emory mouse lenses, and they provided evidence for cataract- and lens-specific up-regulation of a novel receptor tyrosine kinase called ARK. The findings suggest that Emory mouse cataract may be associated with lens- and age-specific increases in the activity of the ARK receptor tyrosine kinase.
AP–1 Regulation of hTTase
Thioltransferase (glutaredoxin), coded in humans by hTTase gene, is a member of oxidoreductases family and participates in oxidative damage repair and thiol/disulfide homeostasis control. Krysan and Lou (p. 1876) provide the first evidence that H2O2–stimulated hTTase gene expression is regulated by AP–1 transcription factor, and mediated through a cellular redox signalling process. This indicates that hTTase is one of many early response genes that cells use to combat oxidative stress.
Differentially Expressed Genes in Pax6 Heterozygous Lenses
Although Pax-6 is a widely studied transcription factor in ocular development and disease, only about two dozen Pax6 target genes are known. Chauhan et al. (p. 1884) examined differentially expressed genes in adult Pax6 heterozygous lenses compared to normal lenses using the RT-PCR differential display and the candidate gene approach. Four functional groups of transcripts: small heat shock proteins (aB-crystallin and Hsp40), crystallins (aB- and bA3/A1- crystallin), transcription factors (Pitx3 and CBP), and components of signal transduction cascades (phosphatase inhibitor protein-1) were found to be genetically downstream of Pax6.
Trk Receptors and RGC Regeneration
There is evidence that neurotrophins are involved in modulating retinal ganglion cell (RGC) survival following optic nerve (ON) lesions. To investigate whether axotomy-induced changes in trk receptor expression accompanies regenerative efforts in RGCs, Cui et al. (p. 1954) studied their expressions on the mRNA and protein levels in the rat retina. They followed optic nerve cut to compare them with the situation after replacement of the ON with an autologous peripheral nerve graft to allow axonal regrowth. The results suggest that differences in changes of trk expression in retina reflect differences in the responses of RGCs to different neurotrophins and indicate that trk receptors are involved in RGC regeneration.
RPE Expression after AV Gene Transfer
Gene transfer into the retinal pigment epithelium could be an attractive strategy to treat neurodegenerative as well as neovascular diseases of the retina. In Kreppel et al. (p. 1965), subretinal injection in rats of low doses of a high-capacity adenoviral vector resulted in efficient transduction of the RPE and in stable transgene expression for over six months. Adverse immune reactions or toxicity were not observed, likely due to the absence of viral gene expression from this vector. Thus, high-capacity adenoviral vectors have a significant potential for the treatment of eye disorders that require durable gene expression.
ABCA4 Mutations in Cone-Rod Dystrophy
It has been shown that mutations in the ABCA4 gene are the major cause for autosomal recessive cone-rod dystrophy (CRD). The present study by Klevering et al. (p. 1980) demonstrates that despite this genetic uniformity the resulting CRD phenotypes are highly heterogeneous. In addition, overlapping features between the various ABCA4-associated retinal disorders, such as Stargardt disease, and CRD are possible. The mere presence of two causative ABCA4 mutations in a CRD patient does not yield enough information to speculate about development of this retinal dystrophy. Future studies will determine the functional consequences of the various ABCA4 mutations, which, in turn, will lead to better predictions of the severity and rate of progression of CRD in a particular patient.
Gene Therapy for Neovascular AMD
Pigment epithelium-derived factor (PEDF) has been demonstrated to promote photoreceptor cell survival and to have antiangiogenic activity. As a single agent with two activities needed in patients with AMD, PEDF has great therapeutic potential. Mori et al. (p. 1994) demonstrate that 4 or 6 weeks after intravitreous or subretinal injection of adeno-associated viral (AAV) vector containing a PEDF expression construct (AAV.PEDF) in mice, there is significant reduction of CNV at sites of laser-induced rupture of Bruch’s membrane. Since AAV vectors mediate long-term transduction of reporter genes in ocular cells, it is expected that intravitreous injection of AAV.PEDF will result in prolonged intraocular expression of PEDF. Provided that necessary safety data are obtained, the effect of intraocular injection of AAV.PEDF should be tested in patients with AMD.
PDGFs Incite TRD in Proliferative Retinopathies
Members of the platelet-derived growth factor (PDGF) family act as stimulatory factors in proliferative retinopathies. It has previously been shown that transgenic mice that carry a rhodopsin promoter/PDGF-A (rho/PDGF-A) transgene have a very mild phenotype with glial epiretinal membranes and no traction retinal detachment (TRD), while mice that carry a rho/PDGF-B transgene develop epiretinal and intraretinal proliferation of vascular and glial cells resulting in TRDs like those seen in patients with vasoproliferative retinopathies. Mori et al. (p. 2001) now show that double transgenics that carry two rho/PDGF-B transgenes or both a rho/PDGF-B and a rho/PDGF-A transgene develop the same phenotype as rho/PDGF-B mice, but over a shorter time period. In contrast, double transgenic mice that carry two rho/PDGF-A transgenes develop prominent glial epiretinal membranes, with no vasoproliferation, resulting in slowly progressive TRDs. These data suggest that blockade of PDGF signaling may help to prevent TRD in either vascular or nonvascular proliferative retinopathies.
RP2 Membrane Targeting Motif
How mutations in RP2 cause retinitis pigmentosa and the function of the protein are unknown. RP2 localizes mainly at the plasma membrane in cultured cells, and a clinically occurring mutation prevents this targeting. RP2 is anchored to the plasma membrane by fatty acids added to the N-terminus of the protein. Chapple et al. (p. 2015) have used mutagenesis to define the precise amino acid motif in RP2 required for this targeting. The targeting motif is highly conserved, further highlighting the importance of these modifications for RP2’s function. Mutations within the motif are predicted to cause disease.
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |