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Related Experiment Video

Updated: Aug 25, 2025

Optimized Minimally Invasive Transscleral Subretinal Injection Technique in Mouse
06:48

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Published on: July 25, 2025

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An Optimized Treatment Protocol for Subretinal Injections Limits Intravitreal Vector Distribution.

Felix F L Reichel1,2, Fabian Wozar1,2, Immanuel Seitz1,2

  • 1University Eye Hospital, Centre for Ophthalmology, University Hospital Tübingen, Tübingen, Germany.

Ophthalmology Science
|October 17, 2022
PubMed
Summary

Subretinal injections (SRis) can lead to adeno-associated virus (AAV) spreading into the vitreous. A simple 3-minute lavage significantly reduces this off-target vector load, enhancing gene therapy safety.

Keywords:
AAVAAV, adeno-associated virusBSS, balanced salt solutionGene therapyIRD, inherited retinal diseaseRetinaSRi, subretinal injectionSubretinal surgeryTIVL, total intravitreal vector loadViral vectorvg, viral genome

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Area of Science:

  • Ophthalmology
  • Gene Therapy
  • Molecular Biology

Background:

  • Subretinal injections (SRis) are crucial for delivering adeno-associated virus (AAV) in retinal gene therapy.
  • Off-target AAV delivery into the vitreous can increase biodistribution and inflammation, potentially compromising safety and efficacy.
  • The intravitreal space is less immune-privileged than the subretinal space, making intravitreal AAV a concern.

Purpose of the Study:

  • To optimize the surgical protocol for subretinal injections (SRis) to minimize off-target adeno-associated virus (AAV) delivery into the vitreous.
  • To reduce the risk of extensive biodistribution and inflammation associated with intravitreal AAV.
  • To enhance the overall safety of retinal gene therapy procedures.

Main Methods:

  • An experimental animal study was conducted using eight cynomolgus monkeys (Macaca fascicularis).
  • Subretinal injections of an AAV2/8 vector were performed at two different viral genome (vg) doses (1x10^11 and 5x10^11 vg).
  • Intravitreal fluid samples were collected post-injection and after a 3-minute balanced salt solution (BSS) lavage to quantify vector genome copies using quantitative polymerase chain reaction.

Main Results:

  • Even uneventful subretinal injections (SRis) resulted in the dissemination of millions of adeno-associated virus (AAV) particles into the vitreous cavity (0.1-0.7% of the total dose).
  • A 3-minute lavage of the intravitreal space with balanced salt solution (BSS) significantly decreased the intravitreal vector load by an average of 96%.
  • Quantitative polymerase chain reaction confirmed a substantial reduction in intravitreal vector genome copies after the lavage procedure.

Conclusions:

  • Minimizing off-target adeno-associated virus (AAV) application after subretinal injection (SRi) reflux is critical for improving retinal gene therapy safety.
  • A simple 3-minute lavage of the intravitreal space effectively reduces the intravitreal vector load.
  • This lavage step should be considered an integral part of surgical protocols for subretinal gene therapy to mitigate risks associated with intravitreal vector dissemination.