Efficacy and dynamics of self-targeting CRISPR/Cas constructs in the retina

Li F, Hung SS, Wang S, Lim JK, Wang JH, Tu L, Daniszewski M, Lo C, Wong RC, Pébay A, King AE, Bui BV, Liu GS, Hewitt AW.

Delivery of CRISPR/Cas proteins remains one of the major remaining impediments to the widespread application of in vivo gene editing and the anticipatory cures of monogenic retinal diseases.  We recently reported the utility of viral mediated CRISPR/Cas gene editing in the retina; however, with such a viral delivery system, active exogenous endonucleases will be maintained in the retina for an extended period. Consequently, CRISPR/Cas genotoxicity is a significant consideration in clinical applications. To address this issue, we have rationally designed a self-destructing “kamikaze” CRISPR/Cas system that disrupts the CRISPR/Cas enzyme itself after active protein expressed. Four different guide RNAs (sgRNAs) were designed to target Streptococcus pyogenes Cas9 (SpCas) and after in situ validation, selected sgRNAs were cloned into the pX552-YFP-sgRNA plasmid to generate yellow fluorescent protein (YFP) targeting “kamikaze-CRISPR/Cas” vector (pX552-YFP sgRNA-SpCas9 sgRNA). The editing efficacy our “YFP targeting kamikaze-CRISPR/Cas” constructs were further validated in vitro in YFP expressed HEK293 cells by Western blot and FACS analysis. Constructs were packaged into an adeno-associated virus 2 (AAV2) vector and administered intravitreally in the Thy1-YFP transgenic mouse and the expression of spCas9 as well as number of YFP fluorescent cells in the retinas were evaluated at 8 weeks after injection. AAV2-mediated LacZ or single YFP targeting SpCas9 sgRNAs were used as controls. Marked reduction of SpCas9 protein concentration (90% at day 2) as well as YFP expression (~66% to 80% at day 10) was found following transfection of our YFP targeting kamikaze-CRISPR/Cas vector in YFP-expressing HEK293 cells compared to the LacZ targeting CRISPR/Cas control. AAV2-mediated in vivo delivery of this construct significantly reduced the number of YFP fluorescent cells (~86% reduction) in the inner retina of Thy1-YFP transgenic mice. Moreover, a significantly decrease of spCas9 mRNA was also detected in AAV2-mediated YFP targeting kamikaze-CRISPR/Cas infected retina over relative to those treated with YFP targeting CRISPR/Cas alone. CRISPR/Cas genotoxicity is a significant consideration in future clinical applications. Our work has demonstrated that a “self-destructing/kamikaze” CRISPR/Cas can be used as a safe and robust tool for refined gene editing in the retina.

 

Thrombosed varix of the superior ophthalmic vein causing unilateral proptosis

Dr Chinh Nguyen1, A/Prof  Nitin Verma1

1Royal Hobart Hospital , Hobart, Australia

78 year-old female presented with proptosis, lid chemosis and diplopia on the right eye. The patient never had a history of head trauma or any other significant trauma. Magnetic resonance imaging angiography showed a thrombosed varix of the superior ophthalmic vein, with no evidence of dural arteriovenous fistula or vascular malformation. Digital subtraction angiography (DSA) was performed one week following the initial presentation, and in the mean time the patient was admitted for observation without anticoagulation. DSA found the thrombosed varix to have re-vascularised on its own at day seven of admission. All symptoms subsided after 2 weeks of the intial presentation. This case study demonstrated that an isolated thrombosed varix of an ophthalmic vein may resolve on it’s own, without the need for anticoagulation or surgery.

Assessment of longitudinal changes in rod function in intermediate age-related macular degeneration using a dark- adapted chromatic perimeter

Dr Chinh Nguyen1, Dr Rogan Fraser2, Dr Rose Tan2, Ms Emily Caruso2, Prof Robyn Guymer2, A/Prof Chi Luu2

1Royal Hobart Hospital , Hobart, Australia, 2Centre for Eye Research Australia, Melbourne, Australia

Purpose: It is well recognised that rod-mediated dark adaptation is significantly impaired in the early stages of AMD. Consistent with the geographical changes in retinal morphology seen in AMD, our baseline study was the first to demonstrate retinotopic variations in dark adaptation recovery rate and sensitivity in intermediate AMD. Longitudinal changes in dark adaptation in early stages of AMD remained unexplored. Therefore, the purpose of this study is to assess the longitudinal changes in rod-mediated dark adaptation in intermediate AMD, at multiple retinal locations, using dark-adapted chromatic perimeter.

Participants: Twenty participants with intermediate AMD and six age-matched control subjects from our baseline study were recalled for the one-year follow up study.

Methods: participants underwent complete ophthalmoscopic examination, multimodal imaging, and dark adaptation. Eyes were graded for AMD features (drusen, pigmentary changes, late AMD), in reference to the Beckman’s classification (defined as drusen ≥ 125 μm with or without pigmentary changes). One eye was designated as the study eye for DA testing.

Main outcome measures: The primary outcomes of this study were retinal sensitivity at 20 minutes and rod criterion time (RCT), which is defined as the time required for sensitivity to recover to a threshold of – 3.0 log unit stimulus intensity. Seven retinal locations were assessed for each patient.

Results: a total of 20 study eyes from 20 participants with intermediate AMD (mean age 72.0 ± 7.0 years) and 6 age-matched control eyes were analysed. There was no significant difference in visual acuity and clinically evident retinal structural changes in our study subjects at baseline and follow-up, aside from one participant who progressed to late AMD. Compared to baseline, progression of DA impairment was not seen in the control group at the one-year follow up. In contrast, both retinal sensitivity at 20 minutes and RCT were further compromised in AMD subjects, particularly in eyes with RPD. Retinotopic variation was once again demonstrated, with the most DA impairment seen within 6 degree eccentricity.

Conclusion: it is feasible to use DAC perimeter to detect longitudinal changes in rod mediated DA in patients with intermediate AMD, at one year follow up and at multiple retinal locations. We demonstrated an overall progression of DA impairment in both static and dynamic rod function at one year follow up in patients with intermediate AMD and more so in eyes with RPD, particularly within the central 6 degree retina.

To Be or not to TB…

Dr William Yates1, Professor  Peter McCluskey2, Doctor  Tani Brown1

1Sydney Eye Hospital, Sydney, Australia,

2Save Sight Institute , Sydney, Australia

 

23y.o student referred in by optometrist with left eye inferior vein occlusion.
Reduced vision over the prior 2-3 weeks.
On examination had a left interior branch vein occlusion with neovascularisation. Evidence of occlusive retinitis on both examination and imaging.
Referred to the uveitis clinic for investigation and management.
Required investigation for causes of occlusive retinitis.
Treatment commenced for both BRVO including anti-VEGF as panretinal photocoagulation, and “presumed TB”.

(Please note the clinical history and findings with subsequent management will be explored further during the presentation)


Biography:

Will is a currently a resident at Sydney Eye Hospital with particular interest in the eye manifestations of systemic diseases. He had conducted research with the Uveitis Service at both Sydney Eye Hospital and the Ocular Inflammatory Group at UNSW

Ocular Experience from Manchester – the Syphilis Capital of Europe

Dr Jane Wells1,3,4, Dr Claire Wood2, Dr Ashish Sukthankar2, Prof Nicholas Jones1,3

1Manchester Royal Eye Hospital, Manchester, United Kingdom,

2Manchester Royal Infirmary, Manchester, United Kingdom,

3The University of Manchester, Manchester, United Kingdom,

4The Canberra Hospital, Canberra, Australia

 

The British Medical Journal named Manchester the “Syphilis Capital of Europe” back in 2004. To date, the population of Manchester continues to be over-represented in the incidence and prevalence of syphilis, including syphilitic uveitis, compared with the UK population.

This presentation summarises statistical and clinical findings from a retrospective observational case series of patients presenting with ocular syphilis to the tertiary referral subspecialist Uveitis Service at Manchester Royal Eye Hospital between 2002 & 2016.


Biography:

Dr Wells is the Uveitis and Medical Retina Specialist at The Canberra Hospital and Senior Lecturer in Ophthalmology at Flinders University in Adelaide.  Her subspecialty training includes a combined Teaching and Uveitis Fellowship at Flinders Medical Centre in Adelaide and a Senior Clinical Fellowship in Uveitis and Medical Retina at Manchester Royal Eye Hospital.

Iritis- Kids vs Adults

Dr Stephen Hing1

1Park Road Eye, Hurstville, Australia

A case of iritis in a child will be presented to highlight the differences between adult and childhood iritis.


Biography:

Senior staff specialist at the Sydney Children’s Hospitals Westmead Campus

Patient reported outcomes of the Tecnis Symfony extended depth of focus IOL

Dr Simran Mangat1

1Tasmania Eye Clinics, Hobart, Australia

 

Introduction
Presbyopia correcting IOLs are a growing market. There are a multitude of options available to patients and surgeons. The Tecnis Symfony IOL is an extended depth of focus IOL creating just one retinal image along an extended focal point rather than multiple images on the retina. This is reported to significantly reduce dysphotopsia. The IOL has proprietary technology that corrects chromatic aberration negating loss of contrast sensitivity from elongating the focal length.

Methods
Retrospective single surgeon patient reported outcome study. A questionnaire was completed on photic phenomena such as glare, halos, starburst, spectacle independence for distance, reading and intermediate vision. Patient evaluation scores for distance, intermediate and near vision. Patient satisfaction with the IOL and a friends and family test of IOL was sought.

Results
Scores were obtained from 24 eyes of 16 Patients. Some patients had not yet completed surgery on the fellow eye either due to the timeline of this study or that they were not yet having visual problems in the fellow eye and wished to defer surgery Eight patients that had Symfony IOLs implanted in both eyes. 7 out of these 8 patients were 100% spectacle independent. The one patient that was not required glasses for intermediate and distance vision, but not near vision.
Eight patients during the study period had only undergone single eye surgery and had the Tecnis Symfony IOL inserted. Two of these reported spectacle independence 100% of the time, 3 patients 25% of the time and the remaining 3 patients required reading glasses 100% of the time
4 of 16 patients reported photic phenomena with glare the most common issue followed by halos and starburst equally.

Conclusions
The Tecnis Symfony IOL is well liked by patients with minimal side effects. Prompt sequential cataract surgery offers better results regarding spectacle independence, 88% in this small sample. Interestingly 63% of patients who underwent single eye surgery reported having to wear reading glasses only 25% of the time or less suggesting they will improve further once the second eye surgery has been performed.


Biography:

Dr Simran Mangat is a general Ophthalmologist with a subspecialty interest in Oculoplastic and lacrimal surgery

Surgical Outcomes of Trabeculectomy and Glaucoma Drainage Implant for Uveitic Glaucoma In A Tertiary Referral Hospital

Dr George Kong1, Dr Hye Jin Kwon2, Dr Lingwei Tao1, Dr Keith Martin3, Dr Lyndell Lim1, Dr Catherine Green1, Dr Jonathan Ruddle1, Dr Jonathan Crowston1

1Royal Victorian Eye and Ear Hospital, Melbourne, Australia,

2Austin Hospital, Melbourne, Australia,

3Cambridge University Hospital NHS Trust, Cambridge, United Kingdom

 

Background
This study examines surgical outcomes of trabeculectomy and glaucoma device implant (GDI) surgery for uveitic glaucoma and examines factors affecting success rate, in particular the activity of uveitis at the time of surgery and in the post-operative period.

Methods
Retrospective chart review of 82 consecutive cases with uveitic glaucoma that either had trabeculectomy (N=54) or GDI surgery (N=28) performed at the Royal Victorian Eye and Ear Hospital between 1 December 2006 and 30 November 2014. Demographics, baseline ocular characteristics, surgical details, uveitis management details, post-operative management, surgical outcomes and complications were recorded.

Results
The average age of patients was 50.0 ± 21.1 [8-85] years. Average follow up duration was 26.4 ± 21.5 months. Combined cataract surgery was performed in 24% of the trabeculectomy surgeries and none in the GDI group. Anterior uveitis was the most common site of uveitis. 57.1% of the GDI group was secondary procedure having had previously failed trabeculectomy.
Overall qualified success rate of the trabeculectomy group was not statistically different to the GDI group, being 67% and 75% respectively (P=0.60). Kaplan-Meier survival analysis showed no significant difference between the two groups (Log-rank P=0.24). There was no significant difference between surgical success rates of primary GDI and secondary GDI operations. The most common post-operative complication was hypotony occurring in 30% of the trabeculectomy group and 36% in the GDI group (P=0.76). Two cases in the GDI group developed endophthalmitis, with none in the trabeculectomy group.
The proportion of active uveitis at the time of operation was higher in the trabeculectomy group compared to the GDI group (35% vs. 14%, P<0.01). Having active uveitis at the time of surgery was not a significant risk factor for failure for trabeculectomy. Recurrence of uveitis following operation occurred in 61% of the trabeculectomy group and 39% of the GDI group (P=0.356). Recurrence of uveitis was significantly associated with surgical failure in the trabeculectomy group (odds ratio OR 4.8, 95% confidence interval CI 1.4-16.4, P=0.02) but not in the GDI group (OR 3.1, 95% CI 0.5-19.7, P=0.39). Even a single episode of recurrence in uveitis activity in the trabeculectomy group can increase failure rate by 5 fold. Other factors for trabeculectomy failure was bleb leak (P=0.032).

Conclusion
Surgical success rate of GDI was not significantly greater than trabeculectomy for uveitic glaucoma in this study. Regular monitoring and early intensive treatment of ocular inflammation is important for surgical success particularly following trabeculectomy.


Biography:

Dr George Kong is a consultant ophthalmologist working at Royal Victorian Eye and Ear Hospital, Austin Hospital and Monash Hospital in Melbourne.  He has completed his clinical glaucoma fellowship in Melbourne and at Cambridge, UK.  He has a particular interest in the management of uveitic glaucoma.