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Article ; Online: Eyelid nystagmus in a child with cardiofaciocutaneous syndrome associated with BRAF mutation.

Wong, Brittany M / Matsumoto, Joyce H / Pineles, Stacy L

Journal of AAPOS : the official publication of the American Association for Pediatric Ophthalmology and Strabismus

2022 Volume 26, Issue 2, Page(s) 95–97

Abstract: Eyelid nystagmus, thought to reflect midbrain or hindbrain disease, is a rare condition that typically occurs in association with neurologic and neuroophthalmic abnormalities. Cardiofaciocutaneous (CFC) syndrome associated with B-raf protooncogene serine/ ...

Abstract	Eyelid nystagmus, thought to reflect midbrain or hindbrain disease, is a rare condition that typically occurs in association with neurologic and neuroophthalmic abnormalities. Cardiofaciocutaneous (CFC) syndrome associated with B-raf protooncogene serine/threonine kinase (BRAF) mutation is a complex syndrome that is associated with neurologic and neuroophthalmologic abnormalities. We report the case of a 4-year-old girl with CFC syndrome with global developmental delay who was referred for evaluation of alternating exotropia, right-sided dissociated vertical deviation, bilateral upgaze nystagmus, and bilateral eyelid fluttering. Neurological work-up revealed bilateral frontocentral sharp waves without seizure activity on electroencephalogram and mild left-sided cerebral volume loss on magnetic resonance imaging. To our knowledge, this is the first reported case of eyelid nystagmus in CFC syndrome. Eyelid nystagmus warrants neurological evaluation, because it is commonly associated with significant neurological abnormalities.
MeSH term(s)	Child ; Child, Preschool ; Ectodermal Dysplasia ; Eyelids/pathology ; Facies ; Failure to Thrive/complications ; Failure to Thrive/diagnosis ; Failure to Thrive/genetics ; Female ; Heart Defects, Congenital/complications ; Heart Defects, Congenital/diagnosis ; Heart Defects, Congenital/genetics ; Humans ; Mutation ; Nystagmus, Pathologic/diagnosis ; Nystagmus, Pathologic/genetics ; Proto-Oncogene Proteins B-raf/genetics
Chemical Substances	BRAF protein, human (EC 2.7.11.1) ; Proto-Oncogene Proteins B-raf (EC 2.7.11.1)
Language	English
Publishing date	2022-02-09
Publishing country	United States
Document type	Case Reports ; Research Support, Non-U.S. Gov't ; Journal Article
ZDB-ID	1412476-2
ISSN	1528-3933 ; 1091-8531
ISSN (online)	1528-3933
ISSN	1091-8531
DOI	10.1016/j.jaapos.2021.12.004
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Zs.A 5188: Show issues

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Article ; Online: Systemic and ocular outcomes in patients with young-onset type 2 diabetes.

Au, Adrian / Bajar, Bryce T / Wong, Brittany M / Daskivich, Lauren P / Hosseini, Hamid / Prasad, Pradeep S

Journal of diabetes and its complications

2023 Volume 38, Issue 2, Page(s) 108670

Abstract: Purpose: To analyze the systemic and ocular outcomes in patients with young-onset type 2 diabetes (YO-DM2) based on grade of presenting diabetic retinopathy (DR).: Methods: Retrospective cohort study analysis of empaneled patients with type 2 ... ...

Abstract	Purpose: To analyze the systemic and ocular outcomes in patients with young-onset type 2 diabetes (YO-DM2) based on grade of presenting diabetic retinopathy (DR). Methods: Retrospective cohort study analysis of empaneled patients with type 2 diabetes <40 years old with retinopathy screening within the Los Angeles Department of Health Services between 01/01/2017-07/01/2021 were included. Patients were stratified based on presenting severity of DR determined on fundus photographs or clinical examination. Patient's systemic co-morbidities and ocular outcomes were then compared across each group. Procedural (e.g. intravitreal injections) and surgical interventions (e.g. pars plana vitrectomy) were documented as performed by the treating physician. Results: 2795 patients were screened from 12,456 patients diagnosed with diabetes younger than age 40 (22.4 %). Of these, 1496 patients were diagnosed with type 2 DM. 1084 (72.4 %) of patients presented without DR, 307 (20.5 %) presented with non-proliferative diabetic retinopathy (NPDR), and 105 (7.0 %) of patients presented with proliferative diabetic retinopathy (PDR). Increasing presenting diabetic retinopathy severity was associated with longer duration of diabetes, greater systemic comorbidities (e.g. diabetic foot disease, neuropathy, chronic kidney or end stage renal disease), worse baseline and final visual acuity, and required more procedural and surgical interventions. Conclusions: Worse presenting DR severity in patients young-onset type 2 diabetes was associated with greater comorbid systemic and ocular disease with worse visual acuity outcomes. <1 % of patients without diabetic retinopathy or with mild NPDR were likely to progress to PDR. Diabetic kidney disease was an independent risk factor for developing neovascular glaucoma and retinal detachments. Prompt evaluation and intervention in patients with YO-DM2 may help reduce the associated systemic and ocular morbidity.
MeSH term(s)	Humans ; Adult ; Diabetic Retinopathy/diagnosis ; Diabetic Retinopathy/epidemiology ; Diabetic Retinopathy/drug therapy ; Diabetes Mellitus, Type 2/complications ; Diabetes Mellitus, Type 2/epidemiology ; Diabetes Mellitus, Type 2/therapy ; Retrospective Studies ; Eye ; Risk Factors
Language	English
Publishing date	2023-12-22
Publishing country	United States
Document type	Journal Article
ZDB-ID	1105840-7
ISSN	1873-460X ; 1056-8727
ISSN (online)	1873-460X
ISSN	1056-8727
DOI	10.1016/j.jdiacomp.2023.108670
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Zs.A 2225: Show issues

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Article ; Online: Regression of Cystoid Macular Edema Three Weeks After Laser for Retinopathy of Prematurity.

Wong, Brittany M / Chu, Alison / Tsui, Irena

Ophthalmic surgery, lasers & imaging retina

2020 Volume 51, Issue 8, Page(s) 472–475

Abstract: Cystoid macular edema (CME) has been reported in the neonatal period associated with preterm birth; however, its pathogenesis remains unclear and is likely multifactorial. The authors report the case of a preterm infant imaged using optical coherence ... ...

Abstract	Cystoid macular edema (CME) has been reported in the neonatal period associated with preterm birth; however, its pathogenesis remains unclear and is likely multifactorial. The authors report the case of a preterm infant imaged using optical coherence tomography (OCT), which revealed severe CME that regressed after laser treatment. Because laser for retinopathy of prematurity aims to reduce vascular endothelial growth factor (VEGF) levels, this case considers the possibility of VEGF-mediated CME pathogenesis. Further, the authors provide additional evidence of the value of OCT for noninvasive visualization of the preterm retina; the modality provides an effective way to evaluate and monitor progression of CME. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:472-475.].
MeSH term(s)	Disease Progression ; Follow-Up Studies ; Gestational Age ; Humans ; Infant, Newborn ; Macular Edema/diagnosis ; Macular Edema/etiology ; Male ; Prognosis ; Retinopathy of Prematurity/complications ; Retinopathy of Prematurity/diagnosis ; Retinopathy of Prematurity/surgery ; Time Factors ; Tomography, Optical Coherence/methods
Language	English
Publishing date	2020-08-18
Publishing country	United States
Document type	Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2701167-7
ISSN	2325-8179 ; 2325-8160
ISSN (online)	2325-8179
ISSN	2325-8160
DOI	10.3928/23258160-20200804-08
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Zs.A 756: Show issues

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Article ; Online: Fungal Infection After Descemet Membrane Endothelial Keratoplasty: Incidence and Outcomes.

Wong, Brittany M / Bonnet, Clémence / Ghaffari, Reza / Houser, Kourtney / DeMatteo, Jennifer / Lau, Nicola / Aldave, Anthony J

Cornea

2022 Volume 42, Issue 6, Page(s) 687–698

Abstract: Purpose: The aim of the study was to describe the incidence, presentation, management, and outcomes of fungal infection after Descemet membrane endothelial keratoplasty (DMEK).: Methods: Retrospective case series of culture-proven fungal infections ... ...

Abstract	Purpose: The aim of the study was to describe the incidence, presentation, management, and outcomes of fungal infection after Descemet membrane endothelial keratoplasty (DMEK). Methods: Retrospective case series of culture-proven fungal infections after DMEK reported in the literature, directly by surgeons, and to the Eye Bank Association of America from January 1, 2011, to December 31, 2020. Results: The domestic incidence of fungal infections, fungal keratitis, and fungal endophthalmitis after DMEK from 2011 to 2020 was 3.5, 1.3, and 2.2 per 10,000 cases, respectively, with no significant increasing trend. Thirty-four cases were identified, 14 (41.2%) published and 20 (58.8%) unpublished. Donor tissue fungal cultures were performed in 20 of the 34 (58.8%) cases and were positive in 19 of the 20 (95.0%), all but one Candida species. Recipient fungal cultures were performed in 29 of the 34 (85.3%) cases and were positive in 26 of the 29 (89.7%), all but one Candida species. Infection presented a mean of 33 ± 38 days (median 23, range 2-200, outlier 949) after transplantation: 25 (73.5%) with endophthalmitis and 9 (26.5%) with keratitis. Topical, intrastromal, intracameral, intravitreal, or systemic antifungal therapy was used in all 27 eyes with treatment data. Surgical intervention (DMEK explantation or partial removal, repeat endothelial keratoplasty, penetrating keratoplasty, and/or pars plana vitrectomy) was required in 21 of the 27 (77.8%) eyes. The corrected distance visual acuity at the last follow-up was ≥20/40 in 13 of the 27 (48.1%) eyes and counting fingers or worse in 6 of the 27 (22.2%) eyes. Conclusions: Fungal infection is a rare but serious complication of DMEK that results in counting fingers or worse corrected distance visual acuity in nearly a quarter of eyes.
MeSH term(s)	Humans ; Descemet Membrane/surgery ; Descemet Stripping Endothelial Keratoplasty/adverse effects ; Descemet Stripping Endothelial Keratoplasty/methods ; Incidence ; Retrospective Studies ; Keratitis/diagnosis ; Keratitis/drug therapy ; Keratitis/epidemiology ; Eye Infections, Fungal/diagnosis ; Eye Infections, Fungal/drug therapy ; Eye Infections, Fungal/epidemiology ; Endophthalmitis/drug therapy ; Endophthalmitis/epidemiology ; Endophthalmitis/etiology ; Endothelium, Corneal
Language	English
Publishing date	2022-08-24
Publishing country	United States
Document type	Journal Article
ZDB-ID	604826-2
ISSN	1536-4798 ; 0277-3740
ISSN (online)	1536-4798
ISSN	0277-3740
DOI	10.1097/ICO.0000000000003102
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Zs.A 1790: Show issues

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Article: Isolation, culture, and genetic engineering of mammalian primary pigment epithelial cells for non-viral gene therapy

Bascuas, Thais / Kropp, Martina / Harmening, Nina / Wong, Brittany M. / Johnen, Sandra / Izsvák, Zsuzsanna / Thumann, Gabriele

Journal of visualized experiments. 2021 Feb. 26, , no. 168

2021

Abstract: Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional ... ...

Abstract	Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.
Keywords	biopsy ; blindness ; cattle ; cell death ; cornea ; electroporation ; epithelium ; fluorescent antibody technique ; gene therapy ; genes ; macular degeneration ; oxidative stress ; photoreceptors ; protein secretion ; retina ; sowing ; transfection ; trypsin
Language	English
Dates of publication	2021-0226
Size	p. e62145.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/62145
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.

Bascuas, Thais / Kropp, Martina / Harmening, Nina / Wong, Brittany M / Johnen, Sandra / Izsvák, Zsuzsanna / Thumann, Gabriele

Journal of visualized experiments : JoVE

2021 , Issue 168

Abstract	Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.
MeSH term(s)	Animals ; Cattle ; Cell Separation/methods ; Cell Survival ; Cells, Cultured ; Electroporation ; Eye Proteins/genetics ; Eye Proteins/therapeutic use ; Genes, Reporter ; Genetic Engineering ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics ; Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use ; Mammals/metabolism ; Mice ; Nerve Growth Factors/genetics ; Nerve Growth Factors/therapeutic use ; Oxidative Stress/genetics ; Rats ; Retinal Pigment Epithelium/cytology ; Serpins/genetics ; Serpins/therapeutic use ; Swine ; Transfection
Chemical Substances	Eye Proteins ; Nerve Growth Factors ; Serpins ; pigment epithelium-derived factor ; Granulocyte-Macrophage Colony-Stimulating Factor (83869-56-1)
Language	English
Publishing date	2021-02-26
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/62145
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Article ; Online: The source of haemorrhage in traumatic basal subarachnoid haemorrhage.

Wong, Brittany / Ong, Beng Beng / Milne, Nathan

Journal of forensic and legal medicine

2015 Volume 29, Page(s) 18–23

Abstract: Traumatic basal subarachnoid haemorrhage (TBSH) following trauma to the head, face or neck is well-established as a cause of death; however it remains a heavily disputed topic as the site of vascular injury is difficult to identify. Whilst many regions ... ...

Abstract	Traumatic basal subarachnoid haemorrhage (TBSH) following trauma to the head, face or neck is well-established as a cause of death; however it remains a heavily disputed topic as the site of vascular injury is difficult to identify. Whilst many regions within the vasculature of the head and neck have been proposed as more susceptible to rupture, the vertebral artery remains the focal point of many investigations. We present a retrospective case review of TBSH in our forensic centre at Forensic and Scientific Services in Brisbane, Australia, from 2003 to 2011. Thirteen cases of TBSH were found, one case excluded due to vasculopathy. All decedents were male, the majority of which were involved in an altercation receiving blows to the head, face, or neck and were unconscious at the scene. All victims were under the influence of alcohol, drugs, or a combination thereof. External examination revealed injuries to the head, face, and neck in all cases. Various combinations of further examination techniques were used during the post-mortem examination including brain and/or cervical spine retention, CT imaging, and angiography. Vascular injury was identified in eight of the twelve cases, all of which occurred intracranially, with seven involving the vertebral artery. Histology was most reliable in identifying the rupture site and angiography failed to reveal a rupture site. The added benefits of histology over angiography are the ability to identify the microscopic architecture of the tear and to diagnose vasculopathy that may have rendered the individual more susceptible to TBSH.
MeSH term(s)	Adult ; Angiography ; Diagnostic Imaging ; Forensic Pathology ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Rupture ; Subarachnoid Hemorrhage, Traumatic/etiology ; Subarachnoid Hemorrhage, Traumatic/pathology ; Substance-Related Disorders ; Tomography, X-Ray Computed ; Vertebral Artery/injuries ; Vertebral Artery/pathology ; Violence ; Wounds and Injuries/pathology ; Young Adult
Language	English
Publishing date	2015-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2268721-X
ISSN	1878-7487 ; 1752-928X
ISSN (online)	1878-7487
ISSN	1752-928X
DOI	10.1016/j.jflm.2014.09.012
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Zs.A 4217: Show issues

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Article ; Online: Lower limb linear morphoea in a pregnant woman with known Graves' disease and cytomegalovirus immunoglobulin M positivity.

Wong, Brittany / Piliouras, Peter / Mortimore, Rohan / Zonta, Mark / Tucker, Simon

The Australasian journal of dermatology

2015 Volume 56, Issue 4, Page(s) e96–8

Abstract: We report a case of linear morphoea in a 21-year-old woman with known Graves' disease who was also cytomegalovirus (CMV) IgM-positive and in her early first trimester of pregnancy. The histopathology showed hyalinisation of the dermis with perivascular ... ...

Abstract	We report a case of linear morphoea in a 21-year-old woman with known Graves' disease who was also cytomegalovirus (CMV) IgM-positive and in her early first trimester of pregnancy. The histopathology showed hyalinisation of the dermis with perivascular superficial and deep lymphocytic infiltrates extending into the septae of the subcutis and impinging on adjacent lobules; there was also fibrosis of the subcutis. Magnetic resonance imaging showed T2 high intensity of the subcutaneous tissue and intermuscular planes of the thigh and leg. It is well known that morphoea can follow pregnancy and thyroid disease and that CMV is postulated to contribute in some patients. This case highlights a combination of risk factors for this disease that have not been reported collectively in the literature to our knowledge.
MeSH term(s)	Antibodies, Viral/blood ; Cytomegalovirus/immunology ; Female ; Graves Disease/complications ; Humans ; Immunoglobulin M/blood ; Lower Extremity ; Pregnancy ; Pregnancy Complications/pathology ; Scleroderma, Localized/complications ; Scleroderma, Localized/pathology ; Young Adult
Chemical Substances	Antibodies, Viral ; Immunoglobulin M
Language	English
Publishing date	2015-11
Publishing country	Australia
Document type	Case Reports ; Journal Article
ZDB-ID	138052-7
ISSN	1440-0960 ; 0004-8380
ISSN (online)	1440-0960
ISSN	0004-8380
DOI	10.1111/ajd.12173
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Article: Are they safe in there? - patient safety and trainees in the practice.

Byrnes, Patrick D / Crawford, Margaret / Wong, Brittany

Australian family physician

2012 Volume 41, Issue 1-2, Page(s) 26–29

Abstract: Background: Due to the projected increase of medical graduates and general practice registrars, a rapid increase in new trainers and practices is required. The resulting mix of relatively inexperienced trainers and trainees makes the examination of the ... ...

Abstract	Background: Due to the projected increase of medical graduates and general practice registrars, a rapid increase in new trainers and practices is required. The resulting mix of relatively inexperienced trainers and trainees makes the examination of the important question of patient safety even more pertinent. Objective: To describe practical techniques that look beyond the door of the closed consulting room to detect unconscious incompetence in trainees. Discussion: Trainees can both be conscious of their incompetence and ask for help, or unconscious of their incompetence. Many articles have been written on teaching trainees who ask for help, but it is the trainee who does not ask for help who may be at most risk of serious problems, and therefore compromise patient safety. Formative assessment and feedback should be used to empower trainees as self-regulated learners. There are seven principles of good feedback practice that help develop self-regulation. This article provides practical teaching tips for supervisors in general practice.
MeSH term(s)	Clinical Competence ; Education, Medical/methods ; General Practice/education ; Humans ; Patient Safety ; Quality of Health Care
Language	English
Publishing date	2012-01
Publishing country	Australia
Document type	Journal Article
ZDB-ID	423718-3
ISSN	0300-8495
ISSN	0300-8495
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Article ; Online: Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

Yiu, Glenn / Tieu, Eric / Nguyen, Anthony T / Wong, Brittany / Smit-McBride, Zeljka

Investigative ophthalmology & visual science

2016 Volume 57, Issue 13, Page(s) 5490–5497

Abstract: Purpose: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells.: Methods: CRISPR sequences targeting exon 1 of human ... ...

Abstract	Purpose: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. Methods: CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Results: Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. Conclusions: The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.
MeSH term(s)	Bacterial Proteins/genetics ; CRISPR-Associated Protein 9 ; Cells, Cultured ; Choroidal Neovascularization/genetics ; Choroidal Neovascularization/metabolism ; Choroidal Neovascularization/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Humans ; Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Real-Time Polymerase Chain Reaction ; Retinal Pigment Epithelium/cytology ; Retinal Pigment Epithelium/metabolism ; Vascular Endothelial Growth Factor A/biosynthesis ; Vascular Endothelial Growth Factor A/genetics
Chemical Substances	Bacterial Proteins ; RNA, Guide, CRISPR-Cas Systems ; Vascular Endothelial Growth Factor A ; CRISPR-Associated Protein 9 (EC 3.1.-) ; Cas9 endonuclease Streptococcus pyogenes (EC 3.1.-) ; Endonucleases (EC 3.1.-)
Language	English
Publishing date	2016-10-31
Publishing country	United States
Document type	Journal Article
ZDB-ID	391794-0
ISSN	1552-5783 ; 0146-0404
ISSN (online)	1552-5783
ISSN	0146-0404
DOI	10.1167/iovs.16-20296
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