Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs.

The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity.

Sustained secretion of human growth hormone from TheraCyte devices encapsulated with PiggyBac-engineered retinal pigment epithelium cells.

Growth hormone (GH) deficiency is characterized by impaired growth and development, and is currently treated by repeated administration of recombinant human GH (hGH). Encapsulated cell therapy (ECT) may offer a less demanding treatment-strategy for long-term production and release of GH into circulation. We used PiggyBac-based (PB) transposon delivery for engineering retinal pigment epithelial cells (ARPE-19), and tested a series of viral and non-viral promoters as well as codon-optimization to enhance transgene expression. Engineered cells were loaded into TheraCyte macrocapsules and secretion was followed in vitro and in vivo. The cytomegalovirus (CMV) promoter supports strong and persistent transgene expression, and we achieved clonal cell lines secreting over 6 µg hGH/106 cells/day. Codon-optimization of the hGH gene did not improve secretion. ARPE-19 cells endured encapsulation in TheraCyte devices, and resulted in steady hormone release for at least 60 days in vitro. A short-term pilot experiment in immunodeficient SCID mice demonstrated low systemic levels of hGH from a single 40 µL capsule implanted subcutaneously. No significant increase in weight increase or systemic hGH was detected after 23 days in the GH-deficient lit/SCID mouse model using 4.5 µL capsules loaded with the highest secreting clone of ARPE-19 cells. Our results demonstrate that PB-mediated engineering of ARPE-19 is an efficient way to generate hormone secreting cell lines compatible with macroencapsulation, and our CMV-driven expression cassette allows for identification of clones with high level and long-term secretory activity without addition of insulator elements. Our results pave the way for further in vivo studies of encapsulated cell therapy.

VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy.

Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA-targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.

Animal Models of Choroidal Neovascularization: A Systematic Review.

Purpose: Animal models of choroidal neovascularization (CNV) are extensively used to characterize the pathophysiology of chorioretinal diseases with CNV formation and to evaluate novel treatment strategies. This systematic review aims to give a detailed overview of contemporary animal models of CNV. Methods: A systematic search was performed in PubMed and EMBASE from November 20, 2015, to November 20, 2020, for mammalian animal models of CNV. Following inclusion by two investigators, data from the articles were extracted according to a predefined protocol. Results: A total of 380 full articles, representing 409 independent animal models, were included. Mice were by far the most utilized animal (76%) followed by rats and non-human primates. The median age of rodents was 8 weeks but with a wide range. Male animals were used in 44% of the studies, but 32% did not report the sex. CNV was laser induced in 89% of the studies, but only 44% of these reported sufficiently on standard laser parameters. Surprisingly, 28% of the studies did not report a sample size for quantitative CNV evaluation. Less than half of the studies performed quantitative in vivo evaluation, and 73% evaluated CNV quantitatively ex vivo. Both in vivo and ex vivo evaluations were conducted primarily at day 7 and/or day 14. Conclusions: The laser-induced mouse model is the predominant model for experimental CNV. The widespread use of young, healthy male animals may complicate clinical translation, and inadequate reporting challenges reproducibility. Definition and implementation of standardized methodologic and reporting guidelines are attractive.

CRISPR Gene Therapy of the Eye: Targeted Knockout of Vegfa in Mouse Retina by Lentiviral Delivery.

Genome editing and knockout by virus-based delivery of CRISPR/Cas9 may provide a new option to cure inherited and acquired ocular diseases. Here we describe development and application of lentivirus-based delivery vectors enabling knockout of the Vegfa gene. We show that Streptococcus pyogenes (Sp) Cas9 and single-guide RNAs (sgRNAs) delivered by such vectors selectively can ablate the vascular endothelial growth factor A (Vegfa) gene in mouse retina following a single administration. These findings may contribute to the development of a new therapeutic path in the treatment of ocular diseases including exudative age-related macular degeneration (AMD).

Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds.

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

Suppression of Choroidal Neovascularization by AAV-Based Dual-Acting Antiangiogenic Gene Therapy.

Vascular endothelial growth factor A (VEGFA) is involved in the pathogenesis of vasoproliferative retinal diseases, such as exudative age-related macular degeneration (AMD). The objective of this study was to investigate whether dual-acting therapy based on the simultaneous expression of anti-VEGFA microRNAs (miRNAs) and the secreted, antiangiogenic protein pigment endothelial-derived factor (PEDF) delivered by adeno-associated virus (AAV) vectors provides improved protection against choroidal neovascularization (CNV). To investigate this, a multigenic AAV vector allowing retina pigment epithelium (RPE)-specific expression of anti-VEGFA miRNAs and PEDF was engineered. Robust expression of PEDF, driven by the RPE-specific vitelliform macular dystrophy 2 promoter, was observed in human cells and in mouse retina. A significant reduction in CNV was observed in a laser-induced CNV mouse model 57 days post-injection of the AAV5 particles conveying either anti-VEGFA miRNA and PEDF dual therapy or anti-VEGFA miRNA monotherapy. Overall, CNV reduction was most prominent in animals receiving dual-acting therapy. In both cases, the reduction in CNV was accompanied by a significant attenuation of VEGFA. In conclusion, the presented data reveal that gene therapy targeting VEGFA via multigenic AAV vectors displays combined efficacy, suggesting that dual-acting therapy is an important tool in future eye gene therapy for the treatment of neovascular ocular diseases, including AMD.

Dissecting microRNA dysregulation in age-related macular degeneration: new targets for eye gene therapy.

MicroRNAs (miRNAs) are key regulators of gene expression in humans. Overexpression or depletion of individual miRNAs is associated with human disease. Current knowledge suggests that the retina is influenced by miRNAs and that dysregulation of miRNAs as well as alterations in components of the miRNA biogenesis machinery are involved in retinal diseases, including age-related macular degeneration (AMD). Furthermore, recent studies have indicated that the vitreous has a specific panel of circulating miRNAs and that this panel varies according to the specific pathological stress experienced by the retinal cells. MicroRNA (miRNA) profiling indicates subtype-specific miRNA profiles for late-stage AMD highlighting the importance of proper miRNA regulation in AMD. This review will describe the function of important miRNAs involved in inflammation, oxidative stress and pathological neovascularization, the key molecular mechanisms leading to AMD, and focus on dysregulated miRNAs as potential therapeutic targets in AMD.