Nasreen A. Syed

Safety and Efficacy of Subretinal Readministration of a Viral Vector in Large Animals to Treat Congenital Blindness

After successful gene therapy to correct retinal degeneration in one eye, treatment of the second eye is safe and effective—even when immunity to the vector is present—in nonhuman primates and dogs. Both Sides Now Blind and deaf from birth, Helen Keller—whose life is the subject of the drama The Miracle Worker—once remarked that “the world is full of suffering; it is also full of overcoming.” Ms. Keller knew something about rising above one’s circumstances—despite her dual disability, she became a renowned author, activist, and lecturer. Last year, through a ground-breaking clinical trial, a research team from The Children’s Hospital of Philadelphia (CHOP) and the University of Pennsylvania helped a group of children to overcome their near-complete blindness by a different route—a gene therapy regimen that replaced a mutated gene and partially restored visual function in one eye of all 12 patients. Now, this same team of researchers has taken an essential step toward the ability to offer these patients the same sight-restoring treatment for their second eyes. These trial participants had all inherited a specific type of Leber’s congenital amaurosis (LCA), a class of progressive retinal degenerative conditions that can be detected in infancy and results in complete blindness by the age of 40. The loss of photosensitive retinal cells characteristic of this class of diseases stems from mutations in one of 14 genes. The LCA-RPE65 patients in the CHOP trial all had aberrant copies of the RPE65 gene, which encodes an enzyme in the retinal pigment epithelium whose function is a crucial component of the visual cycle. Therefore, surgeons delivered a wild-type copy of RPE65 housed in an adeno-associated virus vector (AAV2-hRPE65v2) into one of the eyes of each of these trial subjects. As one might imagine, the sight-restoring success of this gene therapy trial has patients clamoring for more—more treatment, that is, of their second eyes. But first, researchers needed to assess whether a second treatment would be safe and effective. Specifically, scientists investigated whether subjects who developed neutralizing antibodies (NAbs) directed against the AAV vector used in the first round of gene therapy would benefit from a second round or whether the anti-AAV NAbs would impede gene transfer. This week’s issue of Science Translational Medicine reports the results of Amado et al. on the immunological and functional consequences of serial subretinal readministration of the RPE65-carrying AAV vector. The authors performed their studies in two large animal models—a canine model of LCA-RPE65 and nonhuman primates without eye disease. Both animals sport eyes similar in size to those of humans, and all primates have macula, a specialized region of the retina responsible for central vision. Also, because nonhuman primates can be infected with wild-type AAV and develop antibodies to viral components, these animals likely give researchers a window into the human immune response that manifests upon vector injection. The authors show that the readministration treatment is safe and effective in both models, even in animals that display preexisting immunity to the vector. These results suggest that a patient who has undergone AAV2-hRPE65v2—mediated gene therapy in one eye may indeed enjoy success from a second surgery. Furthermore, scientists had routinely excluded from clinical trials patients who already carried NAbs to AAVs in their sera. And, as Amado et al. also show, this crowd encompasses a substantial portion of the human population, a number that increases with age. Now, LCA-RPE65 patients with antibodies to AAV may be eligible for gene therapy. This study thus represents an essential step in the further translation of gene therapy for LCA—and a reason for celebration in the gene therapy research community. Leber’s congenital amaurosis (LCA) is a group of severe inherited retinal degenerations that are symptomatic in infancy and lead to total blindness in adulthood. Recent clinical trials using recombinant adeno-associated virus serotype 2 (rAAV2) successfully reversed blindness in patients with LCA caused by RPE65 mutations after one subretinal injection. However, it was unclear whether treatment of the second eye in the same manner would be safe and efficacious, given the potential for a complicating immune response after the first injection. Here, we evaluated the immunological and functional consequences of readministration of rAAV2-hRPE65v2 to the contralateral eye using large animal models. Neither RPE65-mutant (affected; RPE65−/−) nor unaffected animals developed antibodies against the transgene product, but all developed neutralizing antibodies against the AAV2 capsid in sera and intraocular fluid after subretinal injection. Cell-mediated immune responses were benign, with only 1 of 10 animals in the study developing a persistent T cell immune response to AAV2, a response that was mediated by CD4+ T cells. Sequential bilateral injection caused minimal inflammation and improved visual function in affected animals. Thus, subretinal readministration of rAAV2 in animals is safe and effective, even in the setting of preexisting immunity to the vector, a parameter that has been used to exclude patients from gene therapy trials.

Evaluation of retinal photoreceptors and pigment epithelium in a female carrier of choroideremia

PURPOSE To clarify the pathogenesis of choroideremia. STUDY DESIGN Human tissue study. TISSUES: Eyes of an 88-year-old symptomatic female carrier of choroideremia (CHM) and six normal, age-matched donors. METHODS The eyes were processed for histopathologic examination, including immunocytochemistry with an antibody against the CHM gene product, REP-1, and retinal cell-specific markers. RESULTS The CHM carrier retina showed patchy degeneration, but the photoreceptor and retinal pigment epithelium (RPE) loss appeared to be independent. The choriocapillaris was normal except where retinal areas were severely degenerate. The CHM gene product, REP-1, was localized to the cytoplasm of rods but not cones. CONCLUSIONS It has generally been considered that photoreceptor degeneration in CHM is secondary to loss of the choriocapillaris or RPE. This study suggests that the rod photoreceptors are a primary site of disease in CHM.

Sebaceous cell carcinoma of the ocular adnexa: clinical presentations, histopathology, and outcomes.

Purpose: To investigate the clinical features, time to diagnosis, histopathology, treatment, and mortality rates of patients with sebaceous cell carcinoma. Methods: This was a retrospective, consecutive series of patients with sebaceous cell carcinoma at a tertiary referral medical center. Between January 1984 and January 2006, 31 patients with sebaceous cell carcinoma were evaluated at the Department of Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics. The main outcome measures were clinical presentations, treatments, reconstructive procedures, and outcomes. Results: Thirty-one patients were diagnosed with sebaceous cell carcinoma of the ocular adnexa on histopathology. Twenty (65%) of the patients were women and 11 were men. The upper eyelid was involved in 18 patients, lower eyelid in 10, both upper and lower eyelids in 1, and caruncle in 2. Twenty-three patients had in situ disease, pagetoid disease, or both. Eight patients reported symptoms for <6 months and 22 had symptoms for ≤12 months before the diagnosis of sebaceous cell carcinoma was made. Local surgical excision of the tumor as initial treatment was performed in 25 patients. Exenteration was the initial surgery performed in 4 patients. Two patients died from metastatic sebaceous cell carcinoma. Conclusions: Sebaceous cell carcinoma has varied presentations and is commonly misdiagnosed. Tumor-related deaths occurred in only 2 patients (6.7%), which is lower than previous reports and may be related to earlier detection or improved surgical excision techniques.

Orbital hemangiopericytoma and solitary fibrous tumor: a morphologic continuum.

Solitary fibrous tumors (SFT) and hemangiopericytomas (HPC) are soft tissue tumors with known histologic and immunohistochemical overlap. A series of these tumors located in the orbit were analyzed in order to determine whether they could be reclassified based on currently recognized histologic criteria. Ten orbital spindle cell lesions, all of which were positive for CD34 antigen, were examined. Diagnostic criteria for SFT included a cytologically bland spindle cell lesion with variable cellularity and focal dense collagenization with diffuse, strong CD34 reactivity, while the criteria for HPC required a more monotonous cellular proliferation without significant variability in cellularity, a “staghorn” vascular pattern, minimal collagenization, and focal or absent CD34 staining. Tumors with typical histologic and immunohistochemical features of HPC or SFT were diagnosed as HPC and SFT, respectively. Those tumors with histologic or antigenic profiles not classic for HPC or SFT were defined as ‘indeterminate.’ Three lesions were classified as SFT and 1 tumor was diagnosed as HPC through use of the above-cited histologic criteria. All lesions showed positive staining of tumor cells with CD34 antigen in varying amounts and were negative for cytokeratin AE 1-3, epithelial membrane antigen, CD68, and Factor XIlla. One solitary fibrous tumor focally stained for S-100 protein and 1 hemangiopericytoma was focally positive for HHF-3 5. Of the 10 analyzed tumors, 6 were classified as ‘indeterminate.’ Furthermore, 1 lesion whose primary histology was that of an SFT recurred 9 years later with an appearance consistent with an ‘indeterminate’ lesion. Our results call into question the present histologic separation of HPC and SFT in the orbit. As in other sites, including deep soft tissue, these data suggest that SFT and HPC are 2 lesions whose morphologic features are best interpreted to exist along a continuum, rather than 2 lesions with distinctly defined histopathology.

Donor-related Candida keratitis after Descemet stripping automated endothelial keratoplasty.

Purpose: To report 2 cases of donor-to-host transmission of Candida albicans interface keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK). Methods: We performed clinical and microbiologic examinations on 2 patients who underwent DSAEK for pseudophakic corneal edema. Results: Two patients underwent uneventful DSAEK surgery using corneal tissue from the same donor. The donor corneoscleral rims were cultured at the time of surgery and both rims subsequently grew C. albicans and Candida glabrata. Approximately 5 weeks after surgery, infiltrates were noted in the DSAEK interface in both of these patients. Despite treatment with antifungal therapy, the Candida keratitis was unable to be controlled medically and required surgical intervention in each case, after which there was no recurrence of infection. Conclusions: Candida interface keratitis can occur after DSAEK. These 2 cases emphasize the importance of donor corneoscleral rim cultures, especially with the increase in lamellar and endothelial keratoplasty, which can make such interface infections more difficult to diagnose and treat. Patients with fungal positive donor corneal rim cultures should be prophylactically treated with antifungal therapy.

Inadvertent Evisceration of Eyes Containing Uveal Melanoma

OBJECTIVES To report an important complication of ocular evisceration therapy for blind, painful eyes that has been unreported in the literature, and to stress the need for careful preoperative evaluation to exclude occult neoplasms prior to therapy. DESIGN Multicenter, retrospective, nonrandomized clinicopathological case series of patients found to have previously unsuspected uveal malignant melanoma during histopathological examination of blind, painful eyes treated by evisceration. RESULTS Histopathological examination of evisceration specimens disclosed previously unsuspected uveal melanoma in 7 patients who were treated for blind, painful eyes. Inflammation caused by necrosis of the tumor and other ocular tissues led to misdiagnosis as endophthalmitis, orbital cellulitis, or idiopathic orbital inflammation in several cases. Preoperative imaging was not performed in 3 cases and failed to detect tumors in the remaining 4 cases. Failure of necrotic tumors to enhance contributed to misdiagnosis. CONCLUSIONS The presence of a malignant intraocular neoplasm should be excluded prior to evisceration of any blind eye or blind, painful eye, particularly with opaque media. Necrosis-related inflammation can confound the clinical diagnosis of occult lesions, as can failure of necrotic tumors to enhance on imaging studies.

Salzmann's Nodular Corneal Degeneration Clinical Characteristics and Surgical Outcomes

Purpose: To characterize the clinical characteristics and surgical outcomes for Salzmanns nodular corneal degeneration (SNCD). Methods: In this retrospective, noncomparative, observational case series, all patients coded with a diagnosis of SNCD between January 1, 1996, and April 30, 2002 were included. Cases whose clinical description did not match the classic description of this disorder were excluded. Clinical characteristics, surgical procedures, and qualitative outcomes were recorded. Results: Among 103 patients diagnosed with SNCD, 93 (152 eyes) met inclusion criteria. Eighty-three patients (89.2%) were women (P < 0.00001), and 59 patients (63.4%) had bilateral disease. A normal age distribution was noted, with a mean age of 54.3 years (median, 53 years; standard deviation = 16.9). Meibomian gland dysfunction was noted in 51 patients (54.8%), contact lens wear in 31 patients (33.3%), peripheral vascularization in 29 patients (31.2%), pterygium in 15 patients (16.1%), keratoconjunctivitis sicca in 9 patients (9.7%), and exposure keratitis in 4 patients (4.3%). Forty-nine eyes (32.2%) of 37 patients (39.8%) required a total of 62 surgical procedures. Impaired vision led to 53 (85.5%) of these procedures and resulted in improved vision in 42 (79.2%) of these cases. Seven eyes (4.6%) underwent surgical intervention for subjective discomfort or contact lens intolerance, and all had improved symptoms at last follow-up. Conclusions: SNCD appears to be a disorder that occurs predominantly in middle-aged women and may be associated with chronic ocular surface inflammation and/or irritation. It is important to diagnose properly because of the good prognosis with medical and surgical therapy.

Uveitis following intravitreal bevacizumab: a non-infectious cluster.

BACKGROUND AND OBJECTIVE In this retrospective case series, the authors report seven cases of bevacizumab-related uveitis that occurred within a 4-month period. PATIENTS AND METHODS Seven eyes of six patients developed non-infectious uveitis following bevacizumab intravitreal injections in a cohort of 978 consecutive bevacizumab injections. RESULTS The mean age of patients was 74.6 years (range: 26 to 92). All patients developed symptom onset within 1 day of injection. Shared signs and symptoms included corneal edema, anterior chamber and vitreous cell, conjunctival injection, ocular pain, and lack of hypopyon. In all patients, visual acuity returned to within one line of baseline acuity. All seven eyes had been previously injected with bevacizumab, with a mean number of antecedent injections of 6.1 (range: 3 to 12). CONCLUSION A cluster of sterile bevacizumab-related uveitic reactions was described in this case series. Acute onset of symptoms, absence of hypopyon, a predominant anterior segment reaction, and prompt improvement on topical steroid therapy are useful clinical features distinguishing this uveitic syndrome from infectious endophthalmitis.

Patterns of Distribution of Oxygen-Binding Globins, Neuroglobin and Cytoglobin in Human Retina

OBJECTIVE To determine the distribution of 2 intracellular oxygen-carrying molecules, neuroglobin (NGB) and cytoglobin (CYGB), in specific retinal cell types of human retinas. METHODS Specific antibodies against NGB and CYGB were used in immunohistochemical studies to examine their distribution patterns in human retinal sections. Double-labeling studies were performed with the anti-NGB and anti-CYGB antibodies along with antibodies against neuronal (microtubule-associated protein 2, class III beta-tubulin [TUJ1], protein kinase C alpha, calretinin) and glial (vimentin, glial fibrillary acid protein) markers. Confocal microscopy was used to examine the retinal sections. RESULTS Immunohistochemical analysis of human retinal tissue showed NGB and CYGB immunoreactivity in the ganglion cell layer, inner nuclear layer, inner and outer plexiform layers, and retinal pigment epithelium. Neuroglobin immunoreactivity was also present in the outer nuclear layer and photoreceptor inner segments. Neuroglobin and CYGB were coexpressed in the neurons in the ganglion cell layer and inner nuclear layer but not within glial cells. CONCLUSION Neuroglobin and CYGB are colocalized within human retinal neurons and retinal pigment epithelium but not within glial cells. Clinical Relevance Our results suggest that NGB and CYGB may serve a neuroprotective role as scavengers of reactive oxygen species and therefore should be considered when developing therapeutic strategies for treatment of hypoxia-related ocular diseases.

Refractory giant cell arteritis with spinal cord infarction

We report an elderly patient with aggressive steroid-refractory giant cell arteritis manifesting as myelopathy and bilateral visual loss while on treatment. Pathologically, spinal cord infarction was observed and was due to extensive necrotizing granulomatous arteritis of spinal arteries. Spinal cord damage in giant cell arteritis is rare. One prior autopsy report of spinal cord infarction in giant cell arteritis did not identify vasculitic changes in the spinal arteries.