Long-Term Preservation of Cones and Improvement in Visual Function Following Gene Therapy in a Mouse Model of Leber Congenital Amaurosis Caused by Guanylate Cyclase-1 Deficiency

Leber congenital amaurosis (LCA) is a severe retinal dystrophy manifesting from early infancy as poor vision or blindness. Loss-of-function mutations in GUCY2D cause LCA1 and are one of the most common causes of LCA, accounting for 20% of all cases. Human GUCY2D and mouse Gucy2e genes encode guanylate cyclase-1 (GC), which is responsible for restoring the dark state in photoreceptors after light exposure. The Glicy2e(-/-) mouse shows partially diminished rod function, but an absence of cone function before degeneration. Although the cones appear morphologically normal, they exhibit mislocalization of proteins involved in phototransduction. In this study we tested the efficacy of an rAAV2/8 vector containing the human rhodopsin kinase promoter and the human GUCY2D gene. Following subretinal delivery of the vector in Glicy2e(-/-) mice, GC1 protein was detected in the rod and cone outer segments, and in transduced areas of retina cone transducin was appropriately localized to cone outer segments. Moreover, we observed a dose-dependent restoration of rod and cone function and an improvement in visual behavior of the treated mice. Most importantly, cone preservation was observed in transduced areas up to 6 months post injection. To date, this is the most effective rescue of the Glicy2e(-/-) mouse model of LCA and we propose that a vector, similar to the one used in this study, could be suitable for use in a clinical trial of gene therapy for LCA1

Synthesis and Immunological Evaluation of Mannosylated Desmuramyl Dipeptides Modified by Lipophilic Triazole Substituents

Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP) is the smallest pep- tidoglycan fragment able to trigger an immune response by activating the NOD2 receptor. Structural modification of MDP can lead to analogues with improved immunostimulating properties. The aim of this work was to prepare mannosylated desmuramyl peptides (ManDMP) containing lipophilic triazole substituents to study their immunomodulating activities in vivo. The adjuvant activity of the prepared compounds was evaluated in the mouse model using ovalbumin as an antigen and compared to the MDP and referent adjuvant ManDMPTAd. The obtained results confirm that the α-position of D-isoGln is the best position for the attachment of lipophilic substituents, especially adamantylethyl triazole. Compound 6c exhibited the strongest adjuvant activity, comparable to the MDP and better than referent ManDMPTAd

Dominant Cone-Rod Dystrophy: A Mouse Model Generated by Gene Targeting of the GCAP1/Guca1a Gene

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions

Deletion at 14q22-23 indicates a contiguous gene syndrome comprising anophthalmia, pituitary hypoplasia, and ear anomalies

Anophthalmia and pituitary gland hypoplasia are both debilitating conditions where the underlying genetic defect is unknown in the majority of cases. We identified a patient with bilateral anophthalmia and absence of the optic nerves, chiasm and tracts, as well as pituitary gland hypoplasia and ear anomalies with a de novo apparently balanced chromosomal translocation, 46,XY,t(3;14)(q28;q23.2). Translocation breakpoint analysis using FISH and high-resolution microarray comparative genomic hybridization (CGH) has identified a 9.66 Mb deleted region on the long arm of chromosome 14 which includes the genes BMP4, OTX2, RTN1, SIX6, SIX1, and SIX4. Three other patients with interstitial deletions involving 14q22-23 have been described, all with bilateral anophthalmia, pituitary abnormalities, ear anomalies, and a facial phenotype similar to our patient. OTX2 is involved in ocular developmental defects, and the severity of the ocular phenotype in our patient and the other 14q22-23 deletion patients, suggests this genomic region harbors other gene/s involved in ocular development. BMP4 haploinsufficiency is predicted to contribute to the ocular phenotype on the basis of its expression pattern and observed murine mutant phenotypes. In addition, deletion of BMP4 and SIX6 is likely to contribute to the abnormal pituitary development, and SIX1 deletion may contribute to the ear and other craniofacial features. This indicates that contiguous gene deletion may contribute to the phenotypic features in the 14q22-23 deletion patients.8 page(s

Novel SOX2 partner-factor domain mutation in a four-generation family

Anophthalmia (no eye), microphthalmia (small eye) and associated ocular developmental anomalies cause significant visual handicap. In most cases the underlying genetic cause is unknown, but mutations in some genes, such as SOX2, cause ocular developmental defects, particularly anophthalmia, in a subset of patients. Here, we describe a four-generation family with a p.Asp123Gly mutation in the highly conserved partner-factor interaction region of the SOX2 protein, which is important for cell-specific actions of SOX2. The proband in this family has bilateral anophthalmia and several other family members have milder ocular phenotypes, including typical optic fissure coloboma. Expression studies indicate that Sox2 is expressed in the eye at the site of closure of the optic fissure during development. The SOX2 mutation in this family implicates the partner-factor interaction region of SOX2 in contributing to the specificity of SOX2 action in optic fissure closure. Our findings indicate that investigation of SOX2 in a broad range of eye anomaly patients aids in the determination of particular functions of SOX2 in development

E155G mutation in GCAP1 causes photoreceptor degeneration.

<p>Photomicrographs of resin-embedded sections from (<b>a</b>) wild-type (<b>b</b>) <i>Guca1a</i>^+/COD3, and (<b>c</b>) <i>Guca1a</i>^COD3/COD3 mice at five months of age. There is a marked thinning of the outer nuclear layer (ONL) in mutant eyes as shown by the loss of photoreceptor nuclei. There is also an apparent shortening of outer segment length, particularly in the <i>Guca1a</i>^COD3/COD3 homozygous mice. Photoreceptor loss in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice was quantified by counting photoreceptor nuclei in photomicrographs of sections from paraffin-embedded eyes taken at fixed positions around the optic nerve. At five months of age (<b>d</b>), there is 30% loss in <i>Guca1a</i>^+/COD3 and a 46% loss in <i>Guca1a</i>^COD3/COD3 mice. At 12 months of age (<b>e</b>), this has progressed to a 32% loss in <i>Guca1a</i>^+/COD3 and a 49% loss in <i>Guca1a</i>^COD3/COD3 mice. * indicates statistical significance at the 1% probability level. Note that in the lower panels of (a), PNA shows non-cone staining above the ONL and INL. INL inner nuclear layer, ONL outer nuclear layer, IS/OS inner segment/outer segment.</p

Mutant GCAP1 leads to progressive loss of cone photoreceptors.

<p>(<b>a</b>) Representative retinal flat mounts taken from 5 month-old mice stained with PNA and anti-cone opsin antibody. There are fewer PNA-positive cone cells present in mutant compared to wild-type retinae, as well as a fewer opsin-expressing cells in mutant versus wild-type retinae. Quantitative analysis shows that there are significantly fewer cone opsin-expressing cells in mutant retinae than PNA-positive cells, although the labelling of both markers is significantly reduced in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice as compared to wild-type littermates. (<b>b</b>) Representative retinal flatmounts taken from 12 month-old mice confirms progression of cone degeneration; in mutant mice, there are fewer opsin-positive cells at 12 months compared to wild-type littermates.</p

Impaired recovery from bright flash of <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p><b>T</b>he a-wave amplitude from a bright test flash, presented at varying times after a conditioning flash, is plotted as a proportion of the amplitude elicited by the conditioning flash. A value of 0 indicates the amplitude from the second flash is the same as that from the first; full recovery from a bright conditioning flash occurs within two seconds in wild-type mice, and the second flash elicits an amplitude half that of the first when the inter-stimulus interval is approximately 1000 ms. This recovery time is extended to 1600 ms in both heterozygous and homozygous knock-in mice, whilst the amplitude from the second flash fails to reach that elicited by the first flash during the time period of recording.</p

Generation of <i>Guca1a</i>^COD3 ‘knock-in’ mice with E155G mutation in GCAP1.

<p>(<b>a</b>) A vector targeting the endogenous <i>Guca1a</i> locus was constructed to include an A-to-G transversion at nucleotide position 19 in exon 4 of <i>Guca1a</i> (red circle), as well as a <i>loxP</i>-flanked (blue arrow heads) neomycin resistance gene within intron 3. Following <i>Cre</i>-mediated excision of the <i>Neo</i> selectable marker, the resulting locus contained the A-to-G change in exon 4 plus a residual 34 bp <i>loxP</i> sequence in intron 3. This was used to distinguish between mutant and native alleles by PCR – the position of the primers used is indicated by the small blue arrows on the <i>Cre</i>-deleted <i>Guca1a</i> locus. (<b>b</b>) PCR amplicons from wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice (lanes 3, 4 and 5 respectively), with 1 kb DNA ladder (lane 1) and no-DNA control (lane 2). The wild type allele generates a band at 734 bp whereas the mutant allele generates a 768 bp band which includes the residual intronic <i>loxP</i> sequence. Wild-type mice have therefore a single band at 734 bp, homozygous <i>Guca1a</i>^COD3/COD3 mice have a single band at 768 bp, and heterozygous <i>Guca1a</i>^+/COD3 mice have both bands. (<b>c</b>) Sequence of wild type and targeted <i>Guca1a</i> allele showing A-to-G transversion at nucleotide position 19 of exon 4.</p