Gene therapy in its simplest form introduces genetic material into target cells, via non-viral or viral vehicles, to treat or prevent diseases by correcting or supplementing defective genes. Viral vector-mediated gene therapy has been used in clinical trials to treat cardiovascular, muscular, metabolic, neurological, haematological and ophthalmological diseases as well as infectious disorders and cancers. Some of the most efficient viral vectors to emerge from preclinical and clinical studies are adenovirus vectors, adeno-associated virus (AAV) vectors, retroviral vectors and lentiviral vectors. Today, AAV vectors are well established in clinical trials for in vivo gene therapy. In the eye, AAV gene therapies promise to address visual abnormalities that are not curable with currently available conventional medications. Subretinal (SR) injection of several AAV serotypes is a highly effective method for delivering gene treatments to photoreceptors and the retinal pigment epithelium (RPE) in preclinical rodent models, non-human primates (NHPs), and also humans. It is crucial that there are vectors available that allow for secure and effective gene delivery to different cell types. Various factors influence the development of AAV technology in addition to enhancing the efficacy and specificity of the transduction of different cell types. These include reducing host immune responses, enhancing the safety and effectiveness of AAV delivery in clinical settings, and optimizing AAV production. When SR delivery is considered in the context of an inherited retinal degeneration (IRD) patient’s retina with severe degeneration, it becomes a technically and physiologically challenging route of administration. Furthermore, systemic AAV distribution may provide for non-invasive access to the retina, at least in experimental model systems, but it may also result in the co-transduction of several other tissues/organs. While AAV is one of the primary gene delivery methods utilized in retinal gene therapy, nevertheless, further enhancement of the efficiency, tropism, and safety of these vectors is needed. Indeed, many academic and commercial teams are focused on the optimization of AAV technology.
In a new research paper published in Research Journal Molecular Therapy: Methods & Clinical Development, Trinity College Dublin researchers in Ireland, Dr. Arpad Palfi, Dr. Naomi Chadderton, Dr. Sophia Millington-Ward, Iris Post, Professor Pete Humphries, Dr. Paul Kenna, and Professor G. Jane Farrar recently described the retinal utility of AAV-PHP.eB. This AAV serotype, recently developed by California Institute of Technology scientists Professor Viviana Gradinaru and Professor Benjamin E Deverman, can cross the blood-brain barrier and target neurons with high efficacy in mice. The Trinity researchers compared the tropism and effectiveness of AAV-PHP.eB capsid to intravitreal (IVT) and SR delivery of AAV2/2 and AAV2/8 capsids, respectively, which are both very effective at transducing the retina through these paths.
The Trinity research team used systemic (via tail vein, TV), IVT, and SR injections of AAVPHP.eB encoding enhanced green fluorescent protein (EGFP) with a ubiquitous CMV promoter (AAVPHP.eB-CMV-EGFP) in adult mice. The team found that AAV-PHP.eB efficiently transduces RGCs at the doses employed when given by TV and IVT, and that the proportion of EGFP-positive cells in the ganglion cell layer (GCL) and inner nuclear layer (INL) was similar. However, the distribution and pan-retinal coverage of EGFP-positive cells in the retina were more even with TV administration. TV AAVPHP.eB-CMV-EGFP was particularly effective at transducing horizontal cells. Additionally, the authors used qRTPCR to measure levels of EGFP mRNA expression and discovered that levels were comparable in IVT AAV-PHP.eB-CMV-EGFP and IVT AAV2/2-CMV-EGFP transduced retinas, the latter being the most frequently used AAV serotype for IVT administration. Following SR delivery of AAV-PHP.eB-CMV-EGFP, the team confirmed transduction of rod and cone photoreceptors using ARR3 immunocytochemistry and highlighted key differences between AAV-PHP.eB-CMV-EGFP and AAV2/8, the latter being a benchmark AAV serotype for targeting phptoreceptors. Notably, both EGFP fluorescence intensities and EGFP mRNA expression levels in the photoreceptor layer suggested that AAV-PHP.eB provides superior expression levels; 50-70-times higher compared to the AAV2/8 serotype.
The authors also observed EGFP protein in RPE cells of mouse retinas transduced with SR AAV-PHP.eB-CMV-EGFP and SR AAV2/8-CMV-EGFP, most likely the result of transduction of RPE cells and expression of EGFP there, (as opposed to expression caused by phagocytosis of transduced photoreceptor outer segments). They contend that AAV-PHP.eB also transduces the RPE, as is the case with AAV2/8, which is well documented to do so. In support of this, they found that primary RPE cells were also readily transduced by AAV-PHP.eB-CMV-EGFP in vitro.
The Trinity researchers also looked at how AAV-PHP.eB transduction affected retinal glia cell activation. They discovered that after receiving AAV-PHP.eBCMV-EGFP via SR and IVT, astrocytes, Müller cells, and microglia were moderately activated. According to the authors, AAV-PHP.eB may be tolerated in the retina on par with AAV2/2 and AAV2/8.
In summary, ocular gene therapy is a promising and emerging field with the potential to treat both rare and more common acquired retinal conditions. Dr. Arpad Palfi and colleagues reported the development of AAV-PHP.eB as a versatile and effective AAV serotype for retinal transduction, capable of targeting either the inner or outer retina in mice, depending on the delivery route. AAV-PHP.eB successfully targeted RGCs, photoreceptors, and RPE cells, which are involved in a diverse array of ocular disorders. ‘The high efficacy of AAVPHP.eB suggests that we could use lower doses of AAV in therapeutic protocols, which would aid in enhancing the safety profile of AAV therapies’ says Dr Palfi, first author of the study. This work also reveals that systemic administration of AAV-PHP.eB results in uniform and widespread transduction of RGCs and horizontal cells across the mouse retina. However, as with any delivery vector there is significant work ahead before such a delivery vechicle can reach the clinic. Ultimately, in both preclinical and clinical contexts, AAVPHP.eB may have the ability to compete with and potentially replace some of the present benchmark AAV serotypes. The new study provides important insight into the potential of this novel technology to widen the gene delivery options available for ocular gene therapy.
Palfi A, Chadderton N, Millington-Ward S, Post I, Humphries P, Kenna PF, Farrar GJ. AAV-PHP. eB transduces both the inner and outer retina with high efficacy in mice. Molecular Therapy-Methods & Clinical Development. 2022 Jun 9;25:236-49.