RNA Targeting and Gene Editing Strategies for Transthyretin Amyloidosis

Systemic amyloidosis describes a heterogenous group of diseases characterised by amyloid fibril deposition within the extracellular space of various organs. Amyloid fibrils are formed when soluble precursor proteins misfold into insoluble, protease-resistant beta-pleated sheets that are histologically identified by apple-green birefringence when stained with Congo-Red dye and examined under cross-polarised light. Disease occurs when aggregation of amyloid fibrils within the extracellular matrix is sufficient to disrupt the structure, integrity and function of the affected organ [1, 2].

Transthyretin (TTR) amyloidosis, also known as ATTR amyloidosis, is caused by misfolding of the transthyretin protein. Transthyretin is a tetramer naturally synthesised by the liver, and a physiological transport protein for thyroxine and retinol. Disease occurs when transthyretin misfolds into pathogenic amyloid fibrils [3, 4]. In vitro formation of ATTR fibrils occurs when the tetrameric form of TTR becomes unstable and dissociates into monomers or is enzymatically cleaved. TTR instability appears to be promoted by oxidative modifications, age-related failure of homeostatic mechanisms, metal cations and inherited pathogenic genetic mutations [5].

Historically, ATTR amyloidosis was regarded as a rare disease, but advances in diagnostics, increased awareness, and the increasing perception of ATTR amyloidosis as a treatable disease have resulted in significantly increased diagnoses in recent years. Data from the National Amyloidosis Centre (the single UK centre for diagnosis and monitoring of amyloidosis) demonstrated an exponential increase in diagnoses over the last two decades. The incidence of ATTR amyloidosis increased from < 3% of all amyloidosis cases between 1987 and 2009, to 14% between 2010 and 2015, and 25% between 2011 and 2014 [6]. This trend has been observed in Sweden with the prevalence of ATTR amyloid cardiomyopathy (ATTR-CM) increasing from 1 per 100,000 in 2008, to five per 100,000 in 2018 [7]. Despite these improvements, ATTR amyloidosis still remains an underdiagnosed entity. The true prevalence remains unknown, but with autopsy studies demonstrating ATTR amyloid deposits in 25% of myocardial samples in patients aged ≥ 85 years, it is thought to be significantly higher than that reported in the literature [8].

ATTR amyloidosis may be either wild-type ATTR (ATTRwt), which occurs secondary to an acquired pathogenic process associated with aging, or hereditary ATTR (hATTR or ATTRv) amyloidosis, which occurs secondary to an inherited TTR-gene mutation. Each pathogenic TTR variant is caused by a single nucleotide substitution resulting in a missense mutation that is inherited in an autosomal dominant fashion with variable disease penetrance. The clinical phenotype depends on the underlying pathogenic TTR variant [9]. ATTRwt amyloidosis predominantly affects males and presents in later life [10]. Myocardial ATTR amyloid deposition results in a restrictive cardiomyopathy, characterised by biventricular wall thickening, stiffening of the myocardium, and development of systolic and diastolic dysfunction [11, 12]. Extra-cardiac sites of amyloid infiltration are associated with carpal tunnel syndrome, lumbar spine stenosis and tendinopathies [13‐15]. ATTRv amyloidosis typically presents at a younger age with a mixed phenotype comprising polyneuropathy (ATTR-PN) and/or restrictive cardiomyopathy. ATTRv amyloid more commonly deposits in the nervous system than ATTRwt amyloid, and causes a progressive, length-dependent, sensory-motor peripheral polyneuropathy and/or autonomic neuropathy. Over 130 pathogenic TTR variants have been identified, but a small handful are responsible for the majority of cases [16]. Although there is considerable overlap, there is a strong association between the specific TTR variant, the clinical phenotype, and prognosis. However, phenotypic diversity does occur, not only between different causative TTR variants but also within variants.[17] For example p.(Val50Met), probably the most common disease-causing TTR variant worldwide, can manifest as early- or late-onset disease. Early-onset disease (age < 50 years) is associated with predominant ATTR-PN in the absence of cardiac amyloidosis (CA), while late-onset disease is associated with a mixed phenotype consisting of ATTR-PN and ATTR-CM, known as ATTR-Mixed [18]. In contrast, the p.(Val142Ile) variant, thought to be carried by up to 3–4% of African Americans, is strongly associated with a predominant cardiomyopathy [19]. Although ATTR-PN causes severe disabling symptoms, cardiac involvement is the main driver of mortality [20].

Early diagnosis and initiation of disease-modifying therapy is of paramount importance, and associated with reduced morbidity and mortality. Prior to the development of specific disease-modifying therapies, successful treatment of ATTRv-PN had been achieved through liver transplantation, which suppresses the production of variant TTR, and stops ATTRv fibril deposition in the nerves. Successful outcomes following liver transplantation supported the hypothesis that reducing or halting TTR production could prevent disease progression [5]. The last decade has seen multiple advances in the treatment of ATTR amyloidosis. Therapeutic strategies include targeted stabilisation of the transthyretin tetramer to prevent dissociation into amyloidogenic monomers or cleavage into amyloidogenic fragments [21, 22]. Recent years have also seen the development of agents aimed at reducing TTR production by disrupting the relevant messenger RNA (mRNA) with either small interfering RNA (siRNA) [23] or antisense oligonucleotides (ASO) [24], and an exciting novel approach that aims to halt TTR production through gene editing is being investigated [25]. While the majority of therapeutic strategies are aimed at slowing or preventing amyloid formation, it is noteworthy that monoclonal antibodies that target ATTR fibrils and accelerate their removal from vital organs are at various stages of development. If successful, these immunotherapeutic agents have the potential to facilitate organ recovery [5]. This review will explore in detail the current therapeutic strategies aimed at suppressing TTR production through gene silencing and gene editing, and also provide insights into future perspectives.

siRNA are double-stranded RNA molecules that bind complementary mRNA molecules and cause degradation, hence interfering with gene expression. In order to be effective, siRNAs must be delivered into the cytoplasm of the target cells, while avoiding degradation from circulating nucleases and phagocytes, and renal filtration [26‐28].

Anti-sense oligonucleotides (ASOs) are composed of a single strand, typically comprising 16–20 synthetic nucleotides, which binds to complementary mRNA to induce degradation of the mRNA through a variety of mechanisms, such as a sterically blocking ribosome attachment or promoting endogenous ribonuclease H1-mediated degradation [44‐46]. Unlike siRNAs, ASOs are able to enter cells without the use of a transfection reagent [44, 47].

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) were discovered as part of the archaea and bacterial immune systems [64, 65]. The CRISPR sequences are transcribed into small RNAs capable of guiding the system to complementary DNA sequences. The Cas9 endonuclease subsequently binds the DNA, and cleaves it, knocking out the target gene. Harnessing this naturally occurring biological mechanism as a potential ‘cut and paste’ tool for gene sequences has established CRISPR/Cas9-based in vivo gene editing as an exciting novel therapeutic strategy. Utilisation of modified Cas9 also allows the upregulation of target gene expression. CRISPR-Cas9 technology is being investigated as a potential treatment for multiple gene-mediated disease processes [66].

ATTR amyloidosis is the prototypic model disease for targeted in vivo genome-editing therapy. The TTR protein is coded for by a single gene, and circulating TTR is exclusively synthesised by the liver. Targeted hepatocyte delivery of a gene-editing therapy has the potential to completely ‘switch off’ TTR production, and prevent subsequent formation of pathogenic ATTR amyloid fibrils [67]. siRNA- and ASO-based therapies have demonstrated that TTR knockdown through gene silencing is clinically beneficial in ATTR amyloidosis. Furthermore, there is considerable overlap in the physiological role of TTR as a carrier protein for vitamin A and thyroxine. TTR knockdown does not appear to effect thyroid function and vitamin A supplementation, has thus far prevented any potential clinical sequelae from TTR deficiency [23, 24].

NTLA-2001 is a CRISPR-Cas9-based genome-editing therapy, transported by a lipid nanoparticle mediated delivery system. Following a single administration, NTLA-2001 resulted in significant editing of the mouse TTR-gene and 96% reduction in serum TTR levels that lasted at least 12 months. These results were replicated in various animal models including cynomolgus monkeys and transgenic mice carrying the human p.(Val50Met) variant with no adverse events [66].

A two-part open-label, single-dose, phase 1 multicentre study in ATTRv-PN and ATTR-CM patients is ongoing to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of NTLA-2001, the latter by measurement of serum TTR concentration. Interim results from the dose-ascending phase of the study showed that NTLA-2001 produced a dose-dependent TTR knockdown. Patients were pre-medicated with corticosteroids and antihistamines. The infusion was completed in all patients, without interruption, and few serious adverse events were reported. The most commonly reported treatment-related adverse events were minor infusion-related reactions manifesting as nausea, headache, chills and rhinorrhea. Patients who received therapeutic doses of NTLA-2001 had mean TTR knockdown from baseline of at least 90%, which was sustained through to last follow-up. TTR knockdown with NTLA-2001 is expected to be permanent [25]. The dose expansion phase in both the polyneuropathy and cardiomyopathy arms of the trial is ongoing (Table 1) [68].

In summary, the emergence of novel gene-silencing and gene-editing therapies have rapidly expanded the armamentarium of treatments available for ATTR amyloidosis. Targeting TTR production has resulted in tremendous improvements in patient outcomes. With the results of large-scale clinical trials expected over the next few years, a further expansion of medicines licensed for treatment of ATTR amyloidosis is very likely. Whilst there do not appear to be any clinical consequences in the short to medium term of depletion of circulating TTR, the long-term effects of these drugs both in terms of efficacy and safety remain to be determined.