Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays

Abstract

Inherited retinal dystrophies and optic neuropathies cause chronic disabling loss of visual function. The development of recombinant adeno-associated viral vectors (rAAV) gene therapies in all disease fields have been promising, but the translation to the clinic has been slow. The safety and efficacy profiles of rAAV are linked to the dose of applied vectors. DNA changes in the rAAV gene cassette affect potency, the expression pattern (cell-specificity), and the production yield. Here, we present a library of rAAV vectors and elements that provide a workflow to design novel vectors. We first performed a meta-analysis on recombinant rAAV elements in clinical trials (2007-2020) for ocular gene therapies. We analyzed 33 unique rAAV gene cassettes used in 57 ocular clinical trials. The rAAV gene therapy vectors used six unique capsid variants, 16 different promoters, and six unique polyadenylation sequences. Further, we compiled a list of promoters, enhancers, and other sequences used in current rAAV gene cassettes in preclinical studies. Then, we give an update on pro-viral plasmid backbones used to produce the gene therapy vectors, inverted terminal repeats, production yield, and rAAV safety considerations. Finally, we assess rAAV transgene and bioactivity assays applied to cells or organoids in vitro, explants ex vivo, and clinical studies.

Keywords: Cas9; Keywords. retina; adeno-associated virus (AAV); biological activity assay (BAA); enhancer; polyadenylation; pro-viral plasmid; promoter; retinal pigment epithelium (RPE); transgene expression assay (TEA).

Figure 1

An overview of retinal gene therapy strategies. The effects of the gene variations determine the gene therapy rescue strategy to be applied. Physical DNA delivery includes electroporation, sonoporation, magnetofection, and bioballistic (gene gun) methods. Viruses: AAV, Adeno-associated virus; Ad, Adenovirus; alpha, alphavirus; Epstein-Barr virus (EBV); FV, Foamy virus; HSV, Herpes simplex virus; HIV, Human immunodeficiency virus; VACV, Vaccinia virus. Nucleases: ZNF, Zinc-finger nuclease; TALEN, transcription activator-like effector nuclease; CRISPR/Cas, clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonuclease. Route of administration: i.cranial, intracranial; i.a., intraarterial; i.cam., intracameral; i.m., intramuscular; i.v., i.t., intrathecal; intravenous (e.g., tail vein or facial vein); i.vit, intravitreal; o.g., oral gavage; r.o., retro-orbital; sub.r, subretinal; sub-T, sub-Tenon; sub.con., sub conjunctiva; s.c., subcutaneous.

Figure 2

The development of recombinant AAV vectors targeting ocular diseases. (A) Main location (arrow) of frequent gene products (genes indicated) causing retinal diseases (color), and recombinant adeno-associated viral vectors (rAAV)-gene supplementation therapy genes (in clinical trials; underlined genes). (B) Hypothetical rAAV gene cassette and the corresponding plasmid. AAV5, adeno-associated viral vector serotype 5; BC, bipolar cell; Cone, cone photoreceptor; CC, connecting cilium; GC, ganglion cell; GCL, Ganglion Cell Layer; ILM, Inner Limiting Membrane; IPL, Inner Plexiform Layer; ITR, inverted terminal repeat; KanR, kanamycin resistance gene; LCA, Leber congenital amaurosis; MGC, Müller glial cell; ONL, outer nuclear layer; OPL, Outer Plexiform Layer; PRC IS, photoreceptor inner segment; PRC OS, photoreceptor outer segment; polyA, polyadenylation sequence; PRE, post-transcriptional gene regulatory element; RP, retinitis pigmentosa; RPE, retinal pigment epithelium; rod, rod photoreceptor; TSS, transcription start site.

Figure 3

Development of rAAV therapies over the years. (A) Unique rAAV capsids usage over time (%; y-axis left) and unique rAAV treatments (genes) in clinical trials (number; y-axis right; Total = 33 of 57 rAAV total clinical trials for the retina). (B) Unique rAAV treatments (genes) and their promoters for the retina (2007–2013 vs. 2013–2019). (C) Unique rAAV treatments (genes) and their polyadenylation sequences in the retina (total = 28). (D) Sponsors with unique rAAV, Lentiviral vector, and AON treatments in clinical trials for the retina/choroid (genes; %; Total = 38). bGH, bovine growth hormone; CAG/CMV, ubiquitous promoters; late SV40, late Simian Virus; MG, Müller glial cell; RPE, retinal pigment epithelium; PRC-BiP, photoreceptor-bipolar-specific promoter; PRC, photoreceptor-specific promoter; rb β-globin, rabbit β-globin polyadenylation sequence.

Figure 4

How to assess Inverted Terminal Repeats (ITRs) of rAAV and how they allow concatemerization. (A) Restriction enzyme sites in the AAV serotype 2 ITR in the flop configuration. RBE’/RBE binds Rep68 (RBE, Rep-binding element) and initiates the Rep helicase. The Rep helicase nicks the trs (terminal resolution site). Restriction enzyme recognition site indicated in blue and the actual cut in red. Figure adapted from [238]. (B) ITR structure in the nucleus after second-strand DNA synthesis in dividing cells favoring homologous recombination. Most rAAV-vectors form episomal concatemeric circular double-stranded DNA.

Figure 5

A hypothetical model of the spread of rAAV capsids (serotypes 1, 2, 5 8, and 9) after intravitreal or subretinal injection in disease or non-disease mouse retinas in vivo based on the studies [99,294,304,306,308,309]. RPE, retinal pigment epithelium; OLM, outer limiting membrane, ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer; ILM, Inner Limiting Membrane.

Figure 6

Qualitative assessment of biological relevance and time of assay for retina-specific rAAV potency assay models. Abbreviations: hiPSC, human induced pluripotent stem cell; NHP, non-human primates; RPE, retinal pigment epithelium.