In Vivo–Directed Evolution of a New Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous

Abstract

Inherited retinal degenerative diseases are a clinically promising focus of adeno-associated virus (AAV)-mediated gene therapy. These diseases arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), leading to cell death and structural deterioration. Because current gene delivery methods require an injurious subretinal injection to reach the photoreceptors or RPE and transduce just a fraction of the retina, they are suitable only for the treatment of rare degenerative diseases in which retinal structures remain intact. To address the need for broadly applicable gene delivery approaches, we implemented in vivo-directed evolution to engineer AAV variants that deliver the gene cargo to the outer retina after injection into the eye's easily accessible vitreous humor. This approach has general implications for situations in which dense tissue penetration poses a barrier for gene delivery. A resulting AAV variant mediated widespread delivery to the outer retina and rescued the disease phenotypes of X-linked retinoschisis and Leber's congenital amaurosis in corresponding mouse models. Furthermore, it enabled transduction of primate photoreceptors from the vitreous, expanding its therapeutic promise.

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

“…Several AAVs have been tested for retinal gene delivery via the intravitreal route, and some have evoked safety concerns that have not been fully addressed at this stage. 25,43 For example, a recent study suggested that the engineered AAV variant AAV2-7m8 might be more immunogenic when used at high doses via intravitreal injections. 25 Therefore, we chose AAV2 for its known safety and efficacy in targeting RGCs in NHP studies [27][28][29] and the large body of work using this vector in human clinical trials.…”

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…AAV variants have thereby been obtained which, for example, better penetrate the vitreous humor to reach the retina 7 , or exhibit improved transduction of human hepatocytes 6 or keratinocytes 23 . However, libraries consisting of shuffled capsids typically expand, but do not redirect viral tropism and, in addition, do not allow directing the selection towards a particular chosen receptor.…”

“…The latter approaches, referred here as cell entry targeting, either use adaptors possibly shielding the viral capsid from natural receptor binding and bridging between viral capsid and the chosen target receptor (non-genetic approaches) or permanently change the capsid by genetic engineering 4,5 . As of yet, genetic engineering employing DNA shuffling of capsid-encoding genes from different serotypes and directed evolution as means to select mosaic AAV capsids with novel features has demonstrated its promise as powerful strategy to improve, but not to restrict, gene delivery to a tissue of choice [5][6][7] . Similarly, efforts to redirect tropism by insertion of receptor-targeting peptides or by non-genetic targeting approaches were successful, but have so far not resulted in AAVs that do discriminate between defined cell types as specifically as, for example, antibodies or antibody mimetics can do 4,[8][9][10] .…”

Long-term restoration of visual function in end-stage retinal degeneration using subretinal human melanopsin gene therapy

“…Other studies have used AAV vectors containing a mouse bipolar-cell-specific enhancer to target a melanopsin.mGluR6 chimera (17) or rhodopsin (6) to bipolar cells via intravitreal injection. However, there is variation in anatomy between primates and mouse models (18), and this may render the intravitreal approach less effective in humans. Virions delivered via intravitreal injection are diluted more in primates compared with mice due to the larger volume of the vitreous, reducing the concentration of vector reaching retinal cells.…”