Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery

Abstract

We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3-4 days and in the serum for up to 15-20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases.

Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives

“…25 Additional studies with these two vectors have confirmed that AAV-2/5 transduces both RPE and photoreceptor cells in rat and dog, with photoreceptors being more efficiently transduced than with AAV-2/2. 26 More importantly, this study demonstrated that the type 4 capsid allows exclusive and stable transduction of RPE cells, and that this feature is conserved among rodent, canine and non-human primate models. Similar to the chimeric rAAV-2/5 vector, subretinal injection of a complete rAAV-5/5 vector in mouse shows faster expression kinetics and results in higher transduction efficiency compared with rAAV-2/ 2, 27 with RPE cells and photoreceptors being transduced in both case.…”

“…29 For chimeric vectors rAAV-2/5 and rAAV-2/4, subretinal injection in dogs results in stable expression of GFP for more than 18 months (Rolling et al, data not shown), with the tropism being identical to the one observed in the rat: transduction of RPE and photoreceptor cells with type 5 and exclusive transduction of RPE with type 4. 26 …”

“…In lacrymal and nasal fluids, viral genome was also detected as early as 15 min after injection and up to 4 days postinjection (p.i.). 26 Regarding intravitreal injection, it has been demonstrated that following delivery of rAAV-2/2gfp, high levels of GFP protein persist for at least 6 months in the optic nerves and brains of mice and dogs. 20 In addition, injection of rAAV-2/2lacZ in the vitreous cavity of guinea pigs leads to b-galactosidase expression in fibers, glial cells and blood vessels of the optic nerve.…”

In vivo gene transfer to the rat retina using herpes simplex virus type 1 (HSV-1)-based amplicon vectors

“…rAAV-2/4 allowed exclusive and stable transduction of RPE cells in rodent, canine and non-human primate models. 9 Stable gene transfer into the retina using lentiviral vectors was also reported. Early studies demonstrated conflicting results when determining the tropism of HIV vectors pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G).…”

“…In a rAAV or lentiviral context, both of these promoters allowed stable expression of the transgene. 9,13 The RPE-specific promoter versus the ubiquitous CMV promoter does not prevent the progressive loss of detectable GFP signal in HSV-1 amplicon-transduced retinas. In contrast to lentiviral vectors, which integrate into the chromosome, the HSV-1 amplicon remains episomal.…”