Sheryl brings a wealth of editorial experience to MJH Life Sciences’ Eye Care Network, having engaged with the readers and the greater ophthalmic community of 

for more than 20 years. As Group Editorial Director, Sheryl’s purview extends across the print and digital network of 

Ophthalmology Times, Ophthalmology Times Europe, Modern Retina 

 Prior to ophthalmology, Sheryl previously covered the fields of audiology and confectionery science/technology/manufacturing.

Sheryl is also passionate about the mission, vision and values of Ophthalmic World Leaders (OWL) and recently completed her term on its Board of Directors. She continues to volunteer as Editor-in-Chief of the organization’s 

Magazine. In 2013, she was honored with the group’s inaugural Rising Star Award.

In addition, she has served as a judge for 

 Health Care Heroes Awards program as well as the Jesse H. Neal National Business Journalism Awards.

A novel non-invasive ocular imaging method may be able to detect an early indicator of glaucoma in time to prevent disease progression and vision loss, according to research from the New York Eye and Ear Infirmary of Mount Sinai (NYEE). The study was published in 

The study focused on measuring flavoprotein fluorescence (FPF) in the eye. Mitochondria—which are responsible for generating energy in cells—produce FPF when they are stressed, and levels of FPF are elevated in people with glaucoma compared to those with normal eyes.

Mitochondrial dysfunction in the optic nerve, which sends light signals to the brain and is critical for vision, can eventually lead to loss of cells and tissue damage, causing multiple eye diseases such as glaucoma, and macular degeneration, as well as other retinal damage. This is the first comprehensive study to look at FPF changes in the optic nerves in patients with different stages of glaucoma compared to healthy eyes.

“Glaucoma is difficult to diagnose in early stages, and often physicians agonize to confirm subtle signs of progression in advanced stages. Once structural damage to the optic nerve has occurred, it is currently not possible to reverse. The better we become at identifying early or ongoing degeneration, the more proactive we can be at implementing protective therapy,” says principal investigator Dr Richard B. Rosen, Vice Chair and Director of Ophthalmology Research at NYEE and Chief of the Retina Service for the Mount Sinai Health System. “Our study shows FPF may be useful as an objective measure for predicting glaucoma progression earlier than measuring structural damage, with similar sensitivity to visual field changes but easier and potentially more consistently.”

A team of researchers used the OcuMet Beacon—a fundus camera with special filters that specifically isolates the fluorescence, developed by OcuSciences—to analyse 86 eyes. Fifty of the eyes had glaucoma, based upon thinning of the retinal fibre layer, and 36 had no disease. They found FPF, an indicator of mitochondrial oxidative stress, was significantly higher in glaucoma eyes compared with normal eyes, especially in early-stage glaucoma cases where damage is difficult to detect. Levels of FPF correlated with other means of detecting glaucoma including visual field mean deviation, visual field pattern deviation, and retinal nerve fibre layer thickness.

These results suggest that FPF could be used clinically to reliably and objectively detect metabolic evidence of injury due to glaucoma, limiting the need for frequent visual field testing, the gold standard for measuring visual function. In fact, the researchers say, FPF may be a more accurate measurement, as visual fields have a number of downsides—they are subjective, fluctuate with a patient’s attention, and patients can lose concentration during this assessment.

“Previous studies have demonstrated that when the mitochondrial oxidative stress is relieved by medication or surgery, the flavoprotein fluorescence levels go down. This makes the technique very attractive as a sensitive way of monitoring response to therapy,” explains Dr Rosen. “This measurement could potentially be used as a first-line indicator to monitor of glaucoma progression for the patient and the physician.”

Researchers say their next step is to see if FPF can reliably monitor the effect of therapy for glaucoma patients, to see when treatment has flattened the risk curve of progression in advanced cases, as well as identifying patients who require early glaucoma intervention.

Articles

Flavoprotein fluorescence could serve as a new biomarker, according to a Mount Sinai study. This measurement could potentially be used as a first-line indicator to monitor of glaucoma progression for the patient and the physician.

High-tech, noninvasive imaging identifies early glaucoma

First-ever retinal gene therapy: Does real-world experience back its use?

Voretigene neparvovec (Luxturna, Spark Therapeutics) is the first causal treatment for biallelic 

 mutation-associated retinal disease, which regularly progresses to legal blindness. The one-time gene therapy aims to deliver the correct coding sequence of the human 

 to the retinal pigment epithelium and is performed via subretinal injection following vitrectomy.

The therapy was approved by both the European Medicines Agency (in 2018) and United States Food and Drug Administration (in 2017) after the supporting pivotal Phase III study revealed statistically significant functional vision improvement in patients in terms of increased light sensitivity (FST) as well as improved ability to navigate a mobility course at different levels of environmental illumination (MLMT).

New gene therapies like these raise the hope of treating a previously incurable disease with a favourable side effect profile. However, as with any new therapeutic product, there is limited real-world data, and soit is natural that uncertaintiesregarding the durability and benefit-risk ratio exist.

Recent reports of retinal atrophy development in the postoperative course of the disease have led to concerns that voretigene neparvovec could lead to potentially devastating consequences in the long term. It is therefore necessary to closely follow up treated patients with multimodal imaging approaches in order toassess retinal morphology and gain further knowledge on the factors possibly contributing to atrophy development.

Managing recurrent herpes simplex in patients undergoing keratoconus CXL

Nothing is perfect, and although corneal collagen cross-linking (CXL) to treat keratoconus is safe and stops the progression of disease, infections can occur.

Herpes simplex virus (HSV) keratitis is one possibility, according to Dr David R. Hardten from the Minnesota Eye Consultants in Minnetonka, who suggested that most cases associated with CXL are recurrences.

Dr Hardten addressed the question of what causes HSV keratitis to recur and noted that the traumatic epi (epithelium)-off procedure, use of steroids to reduce haze, and exposure to 30 minutes of ultraviolet light may be the culprits in restimulating the virus in patients with prior exposure. He also explained that with the epi-on procedure, the epithelium is disrupted, which leaves the scene open for an HSV recurrence.

“There are a number of different risk factors that can create that environment in which HSV can flourish,” he said.

Gene fragments may reveal unexplained causes of inherited retinal diseases

Retinal cells in zebrafish with the outer segment, the part of photoreceptor cells responsible for transforming light into nervous signals that enable vision, stained in green at the top of the image. The outer segment is significantly degraded in retinal cells with the Srrm3 gene knocked out (right) compared to normal retinal cells (left). (

Researchers at the Centre for Genomic Regulation (CRG) in Barcelona reveal that 

 is a master regulator gene crucial for the development of photoreceptors, cells in the back of the retina which capture and process light, sending signals to the brain that enable vision. Knocking the gene out in zebrafish resulted in severe visual impairment.

According to a news release from the Centre of Genomic Regulation, the research found that, in vertebrates, 

 works by regulating alternative splicing, a process that allows cells to make more than one type of protein from a single gene and is particularly prominent in neuronal cells. The misregulation of alternative splicing can have a devastating impact on human health, for example in cancer or neurological disorders.

Identifying viral vectors for efficacious gene therapy delivery via computational platform

A novel computational platform developed by researchers from the University of Pittsburgh School of Medicine identifies top-performing viral vectors that could deliver gene therapies to the retina with maximum efficiency and precision.

According to the university, the technology, described in a paper published in the journal 

, streamlines development of gene therapy approaches for the treatment of genetic blinding disorders. The approach saves precious time and resources by speeding up identification of suitable gene-carrying candidates able to deliver therapy to an affected part of the retina with astounding accuracy.

“Vision loss has a huge impact on quality of life. It has long been near the top of the greatest fears of people, alongside cancer and Alzheimer’s disease,” senior author McGowan Institute for Regenerative Medicine affiliated faculty member Dr Leah Byrne, PhD, an assistant professor of ophthalmology at Pitt, said in the university's news release. “But the field of vision restoration has entered a new era, where many patients have received effective treatment for the very first time. Because of that, the potential of our new platform is thrilling—it will allow us to translate emergent therapies that are already working for some patients into the clinic much more rapidly.”

Good vision, minimal disturbances achieved with presbyopia IOL

Bilateral implantation of the Precizon Presbyopic NVA Multifocal IOL (Ophtec) provides “satisfying” uncorrected vision at all distances for patients undergoing cataract surgery or clear lens extraction, according to Dr Mike P. Holzer, an ophthalmologist in private practice in Weinheim, Germany.

The company describes this technology as a hybrid, acrylic IOL with a continuous transitional focus (CTF) optic to correct presbyopia, which according to Dr Holzer is a key feature of this IOL. CTF provides seamless transition from one distance to another. The lens is not currently approved by the FDA.

An end-of-week review of what happened in ophthalmology from 30 July to 5 August.

Weekly review in ophthalmology: 30 July - 5 August

 gene encodes a key enzyme in the retinoid cycleand is responsible for the regeneration of the light-sensitive component of rhodopsin, our visual purple.

 When light enters the eye, it hits the photoreceptors in the retina leading to a conversion of the light signal into a chemical signal.

This so-called photoisomerization of the vitamin A derivative 11-cis-retinal (to all-trans-retinal) cannot take place unlessthere is sufficient functional 11-cis-retinal available. Since 11-cis-retinal decays after initiation of the visual process, it must be perpetually regenerated by specific metabolic processes in the retinal pigment epithelium in order to initiate and maintain the visual process.

 gene result in deficiency or severely functionally impaired isomerohydrolase activity, causing a severe rod-cone dystrophy.

-associated retinal dystrophy are thought to result from different residual activity of the enzyme.

Clinically, the two most common forms of 

-related retinal disease are Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EOSRD). In both forms, visual impairment is first noticed at birth and during the first years of life, respectively, which worsens over time eventually leading to complete blindness.

LCA is considered the most severe form of early childhood blindness and was first described by Theodor Leber in 1869.

 Affected infants usually show lack of eye contact and nystagmus and/or present with conspicuous pressing of their eyeballs with fingers, fists or toys (oculo-digital phenomenon).

Parents may report that their child frequently trips over objects or bumps into obstacles, especially in dim light. EOSRD which manifests after infancy has also a very poor prognosis and, like LCA, usually leads to blindness in the third to fifth decade of life.

To date, it is known that LCA can be caused not only by alterations in the 

 gene, but also by mutations in a further 24 genes (see: 

https://sph.uth.edu/retnet/sum-dis.htm#A-genes

 gene might also manifest in the form of retinopathia pigmentosa (RP20).

In the subtype of RP20, a noticeable deterioration of visual acuity usually occurs in young adulthood or adolescence while concentric loss of visual field is already advanced.

-mutation associated retinal disease is a pronounced night blindness which presents as one of the earliest and most characteristic symptoms of the disease, and a progressive irreversible retinal degeneration.

The night blindness can be explained by the functional impairment of the rods, which are already affected at earliest disease stages. Rods, unlike cones, are completely dependent on 11-cis-retinal regeneration through the retinoid cycle of the retinal pigment epithelium. Cones are less affected in early stages of the disease because they can rely on 11-cis-retinal from other sources, such as Müller cells, which explains their better function in early disease stages.

Gene therapy surgery and mechanism of action

 Principle of application of gene therapy with voretigene neparvovec.

 Mode of action of AAV-mediated gene therapy. (

Abbreviations: AAV, adeno-associated virus; dsDNA, double-stranded DNA

Voretigene neparvovec consists of the capsid of an adeno-associated viral vector serotype 2 (AAV2) containing a correct coding sequence (cDNA) of the human 

 This is provided in the form of frozen concentrate, which must be prepared into a vector solution by trained personnel. 

Subsequently, the therapy is provided in asyringe containing the vector solution which mustbe applied within 4 hours after preparation. Following vitrectomy, delivery of the vector solution is performed by using a small injection cannulaby placing it onto the retina and applying slight pressure to create a retinotomy through which the fluid can pass into the subretinal space (see 

The injection may be performed manually with the help of an assisting surgeon or using a foot-pedal controlled injection device. Patients receive a single dose of 1.5x1011 vector genomes voretigeneneparvovec in each eye; the intended target volume is 300 µl.

The injectionforms one or more fluid-filled bubbles under the retina (blebs) which are reabsorbed within 24–48 hours after subretinal delivery, as the drug is taken up by the target cells, the retinal pigment epithelium. Uptake into the target cells is receptor-mediated.

Once in the nucleus, the single-stranded DNA is transcribed into double-stranded DNA, and the mRNA is subsequently translated in the cytosol into the functional protein, the enzymeisomerohydrolase (illustrated in 

Reports of atrophy development following administration

The clinical studies leading to approval of voretigeneneparvovec showed that there are certain risks associated with the gene therapy procedure. However, most of the treatment-related adverse events were transient and mild. These included elevated intraocular pressure (18%), cataract formation (18%), ocular inflammation (8%), retinal tears (8%), dellen (8%) and retinal deposits (8%).

The previously undescribed complication of chorioretinal atrophy development following treatment with voretigene neparvovec was recently reported by ophthalmologist Dr William S. Gange and colleagues.

 Eighteen eyes of 10 patients developed perifoveal chorioretinal atrophy; in 80% of the cases this was seen bilaterally.

Atrophy was first identifiable anywhere between 1 week and 1 year postoperatively at an average of 4.7 months after treatment (the follow-up period ranged from 4–18 months). In 10 eyes, the atrophy occurred within and outside the area of the subretinal bleb, whereas in seven eyes, it formed exclusively within the bleb’s area.

One eye showed atrophy only outside the bleb area. Despite atrophy development, functional resultsremained stable or improved in the majority of patients. Twelve of 18 eyes showed improved visual acuity (VA), whereas in three of the eyes VA did not change.

VA decreased in a further three eyes. After statistical analysis, no significant change in mean VA was found pre- versus postoperatively (

 = 0.45). While all 13 eyes with reliable Goldmann visuals showed improvement (expansion or gain of isopters),paracentral scotomas caused by the atrophy were seen in three eyes.

Another recent publication reported progressive atrophy development in 13 eyes of eight patients.

 All eyes developed atrophy within the bleb area andthree patients additionally developed atrophic changes outside the bleb.

The mean duration of the patients’ follow-up period was 15.3 months (ranging from 6 to 27 months). First signs of developing atrophy as detected by reduced autofluorescence were identifiable in five of eight eyes as early as 2 weeks after treatment which represented the earliest postoperative visit.

At month 3 following therapy, all 13 eyes showed areas of retinal atrophy. Notably, the atrophy area enlarged over time and in six of seven eyes with existing follow-up data after 1 year, atrophy development progressed even after 1 year. Functional improvement shown by FST and perimetry seemed to be overall stable over the observational period despite atrophy development.

Possible explanations for the development of atrophy include immune reactions against the vector genome (e.g., promotor sequence as the CAG promotor in voretigene neparvovec, the expressed transgene or manufacturing-related impurities) or against the capsid. Manufacturing-related factors could also include subtle deviations in the preparation of the gene therapy shortly before therapy administration at the respective treatment centre.

Surgical factors may also play an important role. Mechanical stress and/or damage at the injection site as well as shear stress within the bleb may directly lead to damage of the retina or may trigger deleterious stress responses. This may be particularly relevant in the setting of 

-related retinal disease and other inherited retinal dystrophies in which dystrophic changes and a more fragile, thinned retina may predispose to damage.

Patient-related factors should be considered as well. Age, gender, stage of the disease and immune status of the individual patient could influence the functional outcome and morphological state of the retina after treatment.

Early detection of inherited retinal diseases (IRD) is becoming increasingly important, as earlier diagnoses enable more timely initiations of therapy, and thus potentially lead to a better prognoses for affected patients. Symptoms such as increased sensitivity to light (photophobia), night blindness or nystagmusmay indicate an IRD and require a thorough ophthalmological work-up within a specialised ophthalmogenetic department. 

If there is reasonable suspicion, molecular genetic testing should be carried out. This is crucial to determine whether therapy with voretigene neparvovec is applicable. Gene therapy with this product must only be performed in individuals with confirmed biallelic 

-mutation-associated retinal dystrophy and sufficient viable retinal cells.

Limited real-world data initiallyconfirmedthe tolerable safety profile seen in marketing authorisation trials.

 However, the recent reports of progressive atrophy development following therapy are undoubtedly concerning.

In summary, currently available data is insufficient to draw definite conclusions about the causes of atrophy development and their functional consequences in the long-term. Experiences of other treatment centres, including reports of the exact surgical procedure and patient details as well as longer follow up periods are necessary to reach a more accurate picture on possible long-term effects of the therapy. This could have important implications for the selection of patients and will help predicting the expectable treatment benefit for eligible patients.

Drs Gerhardt, Michalakis,Rudolph,Priglinger andPriglinger are based at the Department of Ophthalmology, Ludwig-Maximilians-University Munich, Mathildenstraße 8, 80336 München, Germany.

1. Redmond TM, Yu S, Lee E, et al. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nature Genetics. 1998;20:344-351.

2. Palczewski K. G protein–coupled receptor rhodopsin. Annu Rev Biochem. 2006;75:743-767.

3. Kiser PD, Golczak M, Palczewski K. Chemistry of the retinoid (visual) cycle. Chemical Reviews. 2014;114:194-232.

4. Takahashi Y, Chen Y, Moiseyev G, Ma J-x. Two point mutations of RPE65 from patients with retinal dystrophies decrease the stability of RPE65 protein and abolish its isomerohydrolase activity. Journal of Biological Chemistry.2006;281:21820-21826.

5. Weleber RG, Michaelides M, Trzupek KM, et al. The phenotype of Severe Early Childhood Onset Retinal Dystrophy (SECORD) from mutation of RPE65 and differentiation from Leber congenital amaurosis. Invest Ophthalmol Vis Sci.2011;52:292-302.

6. Leber T. Über Retinitis pigmentosa und angeborene Amaurose. Archiv für Ophthalmologie 1869;15:1-25.

7. Morimura H, Fishman GA, Grover SA, et al. Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis. Proceedings of theNational Academy of Sciences.1998;95:3088-3093.

8. Wang J-S, Kefalov VJ. An alternative pathway mediates the mouse and human cone visual cycle. Current Biology.2009;19:1665-1669.

9. Sato S, Frederiksen R, Cornwall MC, Kefalov VJ. The retina visual cycle is driven by cis retinol oxidation in the outer segments of cones. Visual Neuroscience. 2017;34.

10. Das SR, Bhardwaj N, Kjeldbye H, Gouras P. Muller cells of chicken retina synthesize 11-cis-retinol. Biochemical Journal.1992;285:907-913.

11. European Medicine Agency.Summary of Product Characteristics for Luxturna. Accessed 7 June 2022.https://www.ema.europa.eu/en/documents/product-information/luxturna-epar-product-information_en.pdf

12. Russell S, Bennett J, Wellman JA, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. The Lancet. 2017;390:849-860.

13. Maguire AM, Russell S, Wellman JA, et al. Efficacy, safety, and durability of voretigene neparvovec-rzyl in RPE65 mutation–associated inherited retinal dystrophy: results of phase 1 and 3 trials. Ophthalmology.2019;126:1273-1285.

14. Gange WS, Sisk RA, Besirli CG, et al. Perifoveal Chorioretinal Atrophy after Subretinal Voretigene Neparvovec-rzyl for RPE65-Mediated Leber Congenital Amaurosis. Ophthalmol Retina.2022;6:58-64.

15. Reichel FF, Seitz I, Wozar F, et al. Development of retinal atrophy after subretinal gene therapy with voretigene neparvovec. Br J Ophthalmol.Online First. 2022.

16. Deng C, Zhao PY, Branham K, et al. Real-world outcomes of voretigene neparvovec treatment in pediatric patients with RPE65-associated Leber congenital amaurosis. Graefe's Archive for Clinical and Experimental Ophthalmology. 2022;260:1543-1550.

Recent reports of retinal atrophy have raised concerns on potential long-term safety.

First-ever retinal gene therapy: Does real-world experience back its use? 

Although HSV likely cannot be prevented all the time, a few things can be done that may preclude the development of HSV keratitis.

The first is to examine the patient for scarring that may be present from a previous infection or from keratitis associated with contact lens wear. Other options include asking the patient about a history of HSV and looking for evidence of previous atopic disease by comparing the 2 eyes.

In patients with a history of disease, Dr Hardten pre-treats the eyes with an oral antiviral agent such as valacyclovir. This is administered 3 times daily about 1 week before the planned CXL procedure and then postoperatively in a tapering dosage.

“I still perform CXL in these patients,” he said. He estimates the risk in those with recurrent HSV to be similar to that in patients undergoing PRK and recommends performing the procedure because the benefits outweigh the risks. He proceeds by increasing the maintenance dose of the antiviral agent in those patients who had a previous infection.

The most important thing to watch for, he noted, is a postoperative persistent or enlarging epithelial defect. These are characterized by the presence of geographic ulcers that have a broader base than the typical narrower dendrites one would see with HSV in patients not taking steroids. The risks associated with administering an antiviral agent to these patients are very low, he commented.

Dr Hardten advised physicians to proceed with caution.

“I think that CXL is relatively safe,” he explained. “However, there are some potential complications.”

Dr Hardten added that it is important to look for past evidence of HSV keratitis and ask patients about previous infections.

“Consider increasing the maintenance dose if patients are already receiving maintenance antiviral therapy,” he concluded. “Prophylaxis therapy is appropriate if you are suspicious. Be wary of epithelial defects; if they are slow to close, consider geographic ulcers. If healing does not occur in 4 to 5 days, consider that this may be HSV keratitis.”

This article is adapted from Dr Hardten’s presentation at the recent annual meeting of the American Society of Cataract and Refractive Surgery in Washington, DC. He is a consultant to Glaukos.

Several issues may restimulate the virus in those patients with prior exposure.

The lens is available in powers ranging from +1.0 to +35.0 D in 0.5-D increments, with an add-on power of +2.75 D. The lens was evaluated in a multicentre, prospective, open-label, single-arm clinical trial (NCT02409771) that looked at the base 10 logarithm of the minimum angle of resolution (logMAR) corrected and uncorrected visual acuity (VA) scores at distance, intermediate, and near; the manifest refraction spherical equivalent; and the defocus curve. Fifty-nine patients were included in the study. All patients in the study underwent bilateral implantation of the IOL.

The results showed that 84% of the patients were within 0.5 D of emmetropia. The investigators observed a slight hyperopic shift. Therefore the constants for the IOL calculation were adjusted. The mean bilateral uncorrected VAs were 0.0 for distance, 0.14 for intermediate, and near 0.2 for near logMAR.

When asked about visual disturbances, most of the patients reported never or almost never having glare or halos. When these visual disturbances did occur, patients reported that they were not bothersome.

“All surgeries for implantation of the IOL were uneventful. The IOL provided very good [VAs] at all distances from far to near. Low percentages of halo and glare occurred. The Precizon Presbyopic IOL implantation is an effective method to provide good [VA] at all distances in presbyopic and cataract patients,” Dr Holzer concluded.

This article is adapted from Dr Holzer’s presentation at the 2022 American Society of Cataract and Refractive Surgery’s Annual Meeting in Washington, DC. The study he presented was supported by Ophtec BV, Groningen, the Netherlands.

84% of the patients who received a hybrid, acrylic IOL with a continuous transitional focus (CTF) optic to correct presbyopia were within 0.5 D of emmetropia. 

 was found to specifically regulate the splicing of microexons, tiny fragments of DNA that are just 3 to 27 letters long. Despite their small size, the regulation of microexons has been shown to play a critical role in protein and cellular function.

The researchers identified dozens of different microexons that are mainly present in photoreceptors but not in other neurons. A large proportion of these microexons affect the function of around 70 genes important for the development of a photoreceptor’s outer segment, the part of the cell that absorbs light. The findings are published in the 

Proceedings of the National Academy of Sciences

The study reveals a new layer of cellular specialization required for the unique cellular shape and function of retinal cells, one of the most complex and specialized cells in the human body. Because of this complexity, retinal cells depend on many unique genes for their development, any which of can have a disease-causing mutation and result in vision loss.

One of the most common causes of inherited vision loss is retinitis pigmentosa, a genetic disorder for which the molecular mechanisms involved are poorly understood. Between 40 to 50 percent of cases of retinitis pigmentosa have no explanation, meaning they carry mutations in genes yet to be identified. The authors of the study plan on carrying out future studies to assess whether 

 or the microexons involved could explain some of these cases.

 gene has neither been associated with the development of photoreceptor cells nor with the pathogenesis of retinal diseases before,” said Ludovica Ciampi, PhD student at the CRG and first author of the study. “We are already exploring the gene’s role in patients without a genetic diagnosis. If we find cases with mutations in this specific gene, or on any retinal microexons, it could lead to potential new therapeutic strategies to manage the condition.”

According to ICREA Research Professor Manuel Irimia, understanding microexon regulation in specific cell types is key for identifying new therapeutic targets.

“Photoreceptors have unique properties thanks to the regulation of alternative splicing and microexons,” Irimia concluded. “This helps make the cell more specialized but also perhaps more susceptible to genetic diseases. Modulating splicing activity is now possible, so the more intricate biology we uncover, the more likely we are to find therapeutic targets to treat retinal diseases.”

The study is a result of a collaboration between ICREA Research Professors Luis Serrano and Manuel Irimia at the CRG, as well as the Telethon Institute of Genetics and Medicine of Naples, Italy and the University of Zurich in Switzerland. The work is funded by the European Research Council, Spanish Ministry of Science and Innovation and the Generalitat de Catalunya.

1. Ludovica Ciampi, PhD, et. al. Specialization of the photoreceptor transcriptome by Srrm3-dependent microexons is required for outer segment maintenance and vision. 

Proceedings of the National Academy of Sciences.

 Published July 12, 2022. DOI.org/10.1073/pnas.2117090119 

The findings of this study may have revealed the crucial role of gene fragments in retinal development and vision.

McGowan Institute affiliated faculty member Dr José-Alain Sahel chairman and distinguished professor of the Department of Ophthalmology at the University of Pittsburgh School of Medicine, director of the UPMC Eye Centre, and the Eye and Ear Foundation Endowed Chair of Ophthalmology, is a co-author on the study.

Even though blinding genetic disorders that affect the retina are considered rare, approximately 1 in every 3000 people worldwide carries one or more copies of broken genes that cause retinal degeneration and loss of vision, according to the university. For centuries, many people with inherited blindness were all but guaranteed to spend a portion of their lives in darkness.

Now, with several gene therapies already on the market in Europe and the U.S., and dozens more entering clinical trials, hope for people with inherited blindness is within reach, but a key obstacle remains: ensuring that vectors, or inactivated viruses carrying the therapeutic genetic code, enter the exact cells that scientists are targeting. The retina is composed of hundreds of millions of cells that are arranged into a series of layers, so precisely targeting the vector to a specific location within that universe is not a trivial task.

To approach the problem, researchers developed a computational platform called scAAVengr, which uses single-cell RNA sequencing to evaluate quickly and quantitatively—among dozens of options—which adeno-associated virus vector, or AAV, is best suited for the task of delivering a gene therapy to a specific part of the retina.

The traditional approach of evaluating AAVs is painstakingly slow, requiring several years and many experimental animals. It also is not very precise since it doesn’t directly measure if AAVs not only entered the cells but also delivered their gene therapy cargo.

The university noted that in contrast, scAAVengr uses single-cell RNA sequencing, which detects if the cargo arrives at its destination safely. And with scAAVengr, that process takes months, not years.

The platform’s uses aren’t just limited to the retina—the researchers showed that it works just as well for the identification of AAVs that target other tissues, including the brain, heart, and liver, according to the release.

“A rising tide lifts all boats, and we hope that this technology propels gene therapy treatments not just in the field of vision restoration but for other purposes,” Dr Byrne concluded. “Rapidly developing fields of gene editing and optogenetics all rely on efficient gene delivery, so the ability to quickly and strategically choose the delivery vectors would be an exciting leap forward.”

A novel computational platform identifies top-performing viral vectors that could deliver gene therapies to the retina with maximum efficiency and precision.

Today, more than 50% of patients with wet age-related macular degeneration (AMD) are treated off-label with repackaged intravenous bevacizumab (Avastin) that was formulated and packaged for oncologic use. This anti-VEGF chemotherapy agent is approved for systemic administration in multiple cancers but has no approved indications in ophthalmology, despite its widespread use in treating a variety of retinal diseases.

Other anti-VEGF therapies that are US Food and Drug Administration (FDA)-approved specifically to treat retinal diseases—including ranibizumab (Lucentis), aflibercept (Eylea), brolucizumab-dbll (Beovu), and faricimab-svoa (Vabysmo)—are much more expensive than off-label bevacizumab. The result: Many clinicians are forced by insurance companies to use the much-less-costly systemic bevacizumab for many of their patients.

Because bevacizumab is not intended or packaged for intravitreal injection, it must be obtained from compounding pharmacies to be used in the eye. These pharmacies repackage very small quantities of oncologic bevacizumab into syringes for intravitreal injection for conditions such as wet AMD, diabetic macular oedema, and branch retinal vein occlusion.

Although these repackaged bevacizumab syringes appear to satisfy an urgent clinical and financial need for patients with a variety of retinal disorders, they are known to be associated with considerable public health concerns due to the risks posed to patients from compounding pharmacies’ lack of compliance with a variety of FDA requirements. This article will examine some of the dynamics of repackaging bevacizumab into syringes for intravitreal use and suggest a way to eliminate these risks to allow physicians to continue to use bevacizumab for their patients.

What is repackaging, and why does the FDA allow it?

Pharmacy compounding, including repackaging, is permitted under FDA regulations when a physician prescribes medication for an individual patient that is unavailable commercially in the dosage and/or form needed by that individual patient. Examples of compounding include creating liquid formulations of medications that a baby or older patient cannot swallow in solid form. Bulk compounding of medications is not legal if the medication is commercially available (eg, FDA approved) in a form or dosage that is appropriate to treat the patient.

In the case of repackaged systemic bevacizumab, with no approved ophthalmic formulation, the only way for retina specialists to use the drug for intraocular injection is through off-label pharmacy repackaging. Compounding pharmacies aliquot small doses to create prefilled syringes of bevacizumab from a large vial for intraocular injection. Through this mechanism, retina physicians have relied on repackaged systemic bevacizumab for nearly 15 years to provide a cost-effective anti-VEGF therapy to their patients.

Although repackaged bevacizumab has helped millions of patients maintain and even improve vision for a variety of previously blinding retinal disorders, the approach may, nonetheless, unavoidably put patients at risk. This situation is largely due to the process of drawing up multiple syringes from a single vial, as well as to the fact that bevacizumab—developed and packaged for oncologic use—is not formulated to meet the FDA’s specific standards for ophthalmic injectables. The agency’s chemistry manufacturing and controls standards are an integral part of the FDA requirements for any drug approval, and the syringes that contain aliquoted bevacizumab have not undergone this rigorous review and vetting.

Typically, 70 to 80 separate aliquots are taken from one bevacizumab vial and repacked for intraocular use. This repetitive process opens the door to contamination; one of the most serious risks from contamination is bacterial endophthalmitis. Unfortunately, cases of endophthalmitis caused by repackaging of bevacizumab have been reported.

Other problems can also present. The repackaging process relies on human judgment as to the quantity of drug aliquoted into each syringe, and given that only a tiny amount is needed, even a small variation due to human error can significantly affect the actual dosage that a retina patient eventually receives. Studies

 have shown that prefilled syringes vary in terms of how much bevacizumab is provided, with underdosing being common; the impact of underdosing, of course, is that patients may appear not to be responding appropriately to treatment, when in fact they simply may be receiving a subtherapeutic dose of the anti-VEGF medication. 

Other risks derive from lack of FDA-reviewed analytical methods, specifications, drug stability data, unknown quantities of particulates or bacterial endotoxins, unverified FDA-approved pH levels, or even the temperatures at which the syringes are filled, stored, and shipped. Faulty performance along any of these dimensions can damage the molecule and compromise its safety and efficacy.

The syringes used to draw up an aliquot of bevacizumab can also introduce problems, including precipitating agglomerations of the molecule’s proteins such that the osmolarity of the drug solution in the aliquoted vial is compromised.

 Moreover, the prefilled syringes themselves often do not meet the FDA’s standards for ophthalmic injectables, may contain silicone lubricant that can be expelled into the eye and cause permanent visual obstruction and increases in intraocular pressure,

 or are not ophthalmic syringes at all but rather ones created for other uses (such as insulin injection). There are no standards or guidelines for the syringes containing repackaged bevacizumab.

When the FDA approves a drug for commercial marketing, in addition to establishing safety and efficacy, it also reviews and regulates the actual manufacturing process, the specific manufacturing equipment employed, and the packaging that will be used for the finished drug product. Current Good Manufacturing Process, or cGMP, is the global gold standard for pharmaceutical manufacturing and a threshold that must be met to secure FDA approval. The FDA demands that manufacturers of approved medications comply with all cGMP protocols. Repackaging pharmacies may not abide by cGMP standards when individual syringes are drawn for intraocular use.

In the interests of providing their patients with a proven anti-VEGF therapy at a more workable pricepoint than is possible with the currently approved anti-VEGF retinal therapies, physicians have long relied on repackaged bevacizumab from compounding pharmacies—and indeed the molecule has proven itself a valuable therapy for many years. But a solution to the risks that repackaging entails, as delineated above, would be a bevacizumab molecule that is specifically developed and optimized to meet all the FDA’s rigorous requirements for an ophthalmic injectable, manufactured in a cGMP facility, packaged with a vial or syringe specifically designed for ophthalmic use, and introduced into the market via the established supply chain.

Such a product would bring retina physicians and their patients a safe and effective FDA-approved option, using a trusted and well-understood molecule, that mitigates the risks they currently face from repackaged bevacizumab from compounding pharmacies.

Szilárd Kiss, MD, is the Bob and Dolores Hope–Robert M. Ellsworth, MD, Distinguished Associate Professor in Ophthalmology and director of clinical research in ophthalmology at Weill Cornell Medical College in New York, New York. His clinical interests include complex vitreoretinal surgery and treatment of age-related macular degeneration and diabetic retinopathy.

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2. US Food and Drug Administration. FDA alerts health care providers and patients of the nationwide recall of all lots of sterile products distributed by Clinical Specialties Compounding Pharmacy. 

http://www.fda.gov/NewsEvents /Newsroom/PressAnnouncements/ucm345019

3. US Food and Drug Administration. Drugs: FDA alerts health care professionals of infection risk from repackaged Avastin intravitreal injections. 

http://www.fda.gov/Drugs/Drugsafety/ucm270296

4. Sheyman AT, Cohen BZ, Friedman AH, Ackert JM. An outbreak of fungal endophthalmitis after intravitreal injection of compounded combined bevacizumab and triamcinolone. 

2013;131(7):864-869. doi:10.1001/jamaophthalmol.2013.88

5. Hawe A, Hulse WL, Jiskoot W, Forbes RT. Taylor dispersion analysis compared to dynamic light scattering for the size analysis of therapeutic peptides and proteins and their aggregates. 

. 2011;28(9):2302-2310. doi:10.1007/s11095-011-0460-3

6. Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. 

2003;20(9):1325-1336. doi:10.1023/a:1025771421906

7. Mahler HC, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. 

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8. Liu L, Ammar DA, Ross LA, Mandava N, Kahook MY, Carpenter JF. Silicone oil microdroplets and protein aggregates in repackaged bevacizumab and ranibizumab: effects of long-term storage and product mishandling. 

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Although off-label repackaged bevacizumab syringes appear to satisfy an urgent clinical and financial need for patients with a variety of retinal disorders, they are known to be associated with considerable public health concerns due to the risks posed to patients from compounding pharmacies’ lack of compliance with a variety of FDA requirements.

Intravenous bevacizumab repackaged for intravitreal use: Making the sight-saving treatment safer

This series of articles summarises an afternoon of presentations, plus a debate, on the topic of corneal ectasia that was held as part of the 29th Cologne Adventsymposium, a meeting organised by Laserforum e.V. and recently held in Cologne, Germany. Chaired by Drs Omid Kermani and Georg Gerten, the presentations each considered the condition from a different perspective and are available online.

Part 1, published in the June 2022 issue of 

contribution of eye rubbing to the development of keratoconus

. In this second instalment, we look at developments in diagnostics and classification methods for ectatic corneal diseases. This is based on the presentation that ophthalmologist Prof. Renato Ambrósio Jr. gave during the one-day event.

Reviewed by Dr Omid Kermani and Prof. Renato Ambrósio Jr.

There have been many advancements in investigative technologies in corneal refractive screening and diagnostics over the past twenty years. Different multimodal imaging approaches can provide important information on the state of the cornea. In his presentation at the 29th Cologne Adventsymposium, Prof. Ambrósio explained that applications for corneal ectasia include screening and early diagnosis, prognosis, classifying and staging the disease.

He stated that when contemplating treatment, auxiliary tests are relevant for optimising manifest refraction, contact lens fitting, customised surgical planning and for detailed follow-up postsurgery. The data are also crucial for patient orientation, education and treatment guidance.

In diagnosing ectasia, Prof. Ambrósio believes it is important to think ahead. For example, monitoring corneal topography enables subclinical ectasia to be detected prior to the development of slit-lamp signs and loss of distance-corrected vision.

 However, to enhance the sensitivity further, there is a need for more advanced techniques for three-dimensional tomography and biomechanical assessments of the cornea.

Intravenous bevacizumab repackaged for intravitreal use: Making the sight-saving treatment safer

The pathophysiology and pathomorphology of corneal ectasia: Multimodal imaging techniques yield vital screening and diagnostics data

The need for precision management in glaucoma moves field forward