RNA-targeting strategies as a platform for ocular gene therapy

https://doi.org/10.1016/j.preteyeres.2022.101110Get rights and content

Highlights

  • RNA-targeted therapeutics expand the gene therapy toolbox.

  • Clinical trials promise RNA-targeted therapies for eye disease within the decade.

  • Emergence of CRISPR-Cas RNA editing might accelerate gene therapy for blindness.

Abstract

Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.

Introduction

Genetic medicine (or gene therapy) refers to the therapeutic use or manipulation of genes and their expression to ameliorate or cure genetic disease. The development of drugs that target genetic diseases has long been central focus of scientific research. Particularly, the eye has been a leading organ for the development of gene therapies due to being physically separated, easy to access, immune-privileged, and postmitotic. The small, compartmentalised structure of the eye also means there is limited spread to other organs and low dosages can be sufficient for therapeutic benefit. In addition, several non-invasive techniques such as optical coherence tomography (OCT), adaptive optics imaging, microperimetry and electroretinogram (ERG), are available to study structure and function.

Ocular gene therapy began with the seminal antisense oligonucleotide (ASO) therapeutic fomivirsen for cytomegalovirus (CMV) retinitis and has progressed to the recent FDA approval of the first ocular gene therapy voretigene neparvovec-rzyl (Luxturna®) for one form of Leber's Congenital Amaurosis (LCA), delivered in adeno-associated virus (AAV). AAVs represent another significant advance for gene therapy and remain the vector of choice for therapeutic development due to their safety profile and transduction capabilities. Emerging delivery technologies such as lipid nanoparticles are also notable and now expanding the scope of gene delivery to the eye.

In recent times, RNA has garnered much public attention. Besides an eventful history revealing a multitude of functions (Fig. 1), the molecule has become particularly known for its unstable and transient nature. RNA-targeted therapy is therefore proving to be an attractive alternative to traditional genomic therapies, and providing unique opportunities and challenges for therapeutic development (Damase et al., 2021).

In drug development, RNA-targeted strategies are gaining traction for allowing specific and reversible genetic manipulation that is independent of DNA. This avoids permanent changes in host organisms (Pickar-Oliver and Gersbach, 2019). Although protein targeting strategies, such as monoclonal antibodies, are a popular therapeutic approach offering similar advantages, protein therapeutics are limited by ‘druggable’ targets: only 1.5% of the human genome encodes for protein while 70% encodes for non-coding RNAs (ncRNAs). Targeting RNA thus significantly broadens therapeutic targets (Warner et al., 2018). In addition, targets may be specified simply by knowledge of the target RNA sequence. Recognizing these benefits, RNA engineering was recently reported as a promising candidate to become one of the most impactful advances for science in the 21st century (Thavarajah et al., 2021).

ASO and RNA interference (RNAi) are the two strategies that have been clinically employed for RNA-targeted therapeutics. Using these strategies, 12 drugs have been developed and approved to date for various genetic diseases (Table 1) (Winkle et al., 2021). While offering promise, manufacturing drugs based on these strategies continue to be complex and a cautious approach is still required with ASO and RNAi strategies due to off-target effects that may affect other essential pathways. Delivery is also challenging due to poor cellular transduction and cytotoxicity, requiring carrier proteins or chemical modifications for therapeutic development (Roberts et al., 2020).

In 2012, the description of a programmable gene editing platform transformed biotechnology (Jinek et al., 2012). Known as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas), the technology allowed designing RNA strands that could target specific DNA sequences for cleavage. Subsequent research has led to the discovery of exclusively RNA-targeting CRISPR-Cas systems, such as CRISPR-Cas13. While ASOs have not been experimentally compared with CRISPR-Cas13 systems yet (Palaz et al., 2021), these systems have exhibited enhanced efficiency and specificity over RNAi (Abudayyeh et al., 2019; Cox et al., 2017; Zhang et al., 2021b). CRISPR-Cas13 systems have so far been demonstrated for knockdown, multiplexed targeting, base editing, and demethylation applications across genetic and infectious diseases (Chuang et al., 2021b; Cox et al., 2017; Xie et al., 2021). Importantly, CRISPR-Cas13 systems now allow for all these applications to be achieved through delivery from a single-AAV vector.

In this review, we describe emergence of the existing RNA-targeting strategies, their applications in ocular disease and the current challenges. Then, we discuss the emergence of CRISPR-Cas and the recent RNA-targeting CRISPR-Cas systems, their potential in addressing ocular disease and the solutions they offer. Lastly, we look at the considerations for developing and commercialising novel therapeutics to inform future ocular gene therapy efforts.

This review is focused on RNA editing for ocular disease. For a general overview of small molecule, gene or cell therapies for ocular disease, we refer the reader to recent excellent reviews on therapeutics against acquired ocular diseases (Gagliardi et al., 2019; Lin et al., 2020; Tan et al., 2021) and inherited ocular diseases (Britten-Jones et al., 2022; Fenner et al., 2022; Schneider et al., 2021).

Section snippets

A brief history of RNA and its many functions

RNA was discovered in the 1890s, differentiated from DNA through localising in the cytoplasm and containing ribose sugars (Allen, 1941). It was generally thought to only function in ribosomes for the translation of proteins as ribosomal RNAs (rRNA). This understanding transformed after the description of an unstable RNA intermediate that facilitates protein synthesis, now commonly known as messenger RNA (mRNA) (Brenner et al., 1961; Gros et al., 1961). During this time, the discovery of

Antisense oligonucleotides (ASO)

ASOs are short (12–24 nt) single-stranded nucleic acids (DNA or RNA) programmed to bind to specific complementary mRNA targets through Watson-Crick base-pairing for the modulation of gene expression. ASOs function through inhibiting natural gene expression processes, that have been repurposed for therapeutic applications In 1977, translational activity was shown to be inhibited through hybridisation with complementary DNA in a cell-free system (Paterson et al., 1977). Subsequently, virus

RNA interference (RNAi)

RNAi pathways regulate gene expression by the modulation of the stability and translation of mRNA in cells by sequence-specific double stranded RNA. The mechanisms of post-translational gene silencing were described in the nematode worm (Caenorhabditis elegans) in 1998 when the introduction of dsRNA resulted in the silencing of an endogenous gene (Fire et al., 1998), and termed RNAi. Soon, RNAi was developed into one of the most diversely applicable tools (Elbashir et al., 2001), providing

CRISPR-Cas gene editing

While programmable CRISPR-Cas gene editing was described less than a decade ago (Jinek et al., 2012), CRISPR is an ancient adaptive immune mechanism evolved in bacteria and archaea.

In 1987, unique ‘spacer’ sequences flanked by repeat sequences were reported (Ishino et al., 1987). Recognizing these spacers as identical to viral sequences revealed that bacteria derive these spacers directly from the viruses that infect them to develop ‘vaccination cards’ against subsequent infection (Bolotin et

RNA knockdown with CRISPR-Cas13

While limited, studies with CRISPR-Cas13 against ocular disease have shown promise for the development of therapeutics for glaucoma and neovascular disease.

We recently described methods for designing CRISPR-CasRx-based (RfxCas13d) knockdown experiments, and demonstrated knockdown of VEGFA mRNA in vitro using single vector system (Chuang et al., 2021b). In addition, we also showed efficient knockdown in vitro through delivery of pre-sgRNAs as gBlocks™, eliminating the need for cloning.

RNA as a therapeutic target

Targeting RNA has a number of advantages for the development of therapeutics (Table S4). As the majority of strategies such as ASOs, RNAi and CRISPR-Cas13 interact with their targets via Watson-Crick base-pairing, design of these strategies is relatively straightforward. Once RNA target sequences are known, gRNAs or ASOs can be rationally designed, with potential off-target sites predicted and reduced. This also allows for the custom design of therapeutics to target patient specific sequences,

Future directions and conclusions

Currently, gene editing technology is being developed to treat fatal or debilitating diseases in both adults and children (McCaughey et al., 2016; Wang et al., 2017), and many applications in the eye are conceivable. In light of potential permanent off-target effects introduced by DNA targeting systems, how do we proceed without compromising safety? Targeting RNA may be a solution with the range of efficient and precise RNA-targeting systems now available (Damase et al., 2021).

Going forward, we

CRediT authorship contribution statement

Satheesh Kumar: Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing. Lewis E. Fry: Conceptualization, Formal analysis, Data curation, Writing – review & editing. Jiang-Hui Wang: Writing – review & editing. Keith R. Martin: Writing – review & editing. Alex W. Hewitt: Writing – review & editing. Fred K. Chen: Resources, Writing – review & editing. Guei-Sheung Liu: Conceptualization, Formal analysis, Data curation, Validation, Funding acquisition, Writing –

Acknowledgements

The authors have no conflicts of interest to disclose. This work was funded by grants from the National Health and Medical Research Council of Australia (1185600). The Centre for Eye Research Australia receives Operational Infrastructure Support from the Victorian Government.

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