Abstract
Purpose:
Photoreceptor dystrophies lead to reduced vision as the photoreceptors degenerate. Gene therapy using Adeno-associated virus (AAV) has shown to be safe and effective at preserving photoreceptors. This preservation is robust when AAV is delivered subretinally, but inefficient when delivered intravitreally (IVit) even though IVit delivery of AAV offers practical advantages. Of the naturally occurring AAV serotypes, most don't transduce the retina IVit, and of the serotypes that can, transduction is limited to the ganglion cell layer. Efforts are now underway to develop mutant AAVs that are capable of photoreceptor transduction via IVit delivery.
Methods:
We developed mutations on the AAV2 capsid which displayed enhanced transduction of multiple retinal cell layers across several animal models. This mutant was dubbed AAV2.5. Our interest was to determine the mechanism of action for this enhancement. We assessed difference in receptor usage and receptor binding affinity of AAV2 and AAV2.5 in vitro using several cell lines and affinity chromatography. To identify key amino acids involved in the enhancement, we generated chimeric capsid carrying each of the 5 amino acids modified in AAV2.5 separately and produced viral preps carrying reporter gene for testing in vivo. Finally, we tried to promote greater transduction in the retina by mixing soluble heparin with AAV2 and AAV2.5.
Results:
We saw no difference in receptor usage or affinity between AAV2 and AAV2.5, indicating a still unknown mechanism for the enhancement in the retina. Similar to published results in muscle, the threonine insertion found in AA2.5 but absent from AAV2 seemed to be a key amino acid necessary for increased retinal transduction. While soluble heparin mixed with AAV2 showed a greater number of retinal cells being transduced, soluble heparin mixed with AAV2.5 showed no increase in transduction.
Conclusions:
The lack of difference in primary heparin sulfate receptor usage and affinity may indicate that AAV2.5 has a secondary receptor usage or downstream advantages in the viral endocytic pathway. Current work is aimed at determining what mechanisms are at play for enhanced retinal transduction, notably by real time imaging of capsid particle critical steps. Future work will investigate newly identified amino acid insertions in combination with chimeric AAV2.5 to fully exploit maximum robust transduction profile in ocular target tissue.
Keywords: 688 retina •
538 gene transfer/gene therapy •
674 receptors