Drug and Gene Therapy for Treating Variant Transthyretin Amyloidosis (ATTRv) Neuropathy

Abstract

Variant Transthyretin Amyloidosis (ATTRv) neuropathy is an adult-onset, autosomal dominant, lethal, multisystemic disease due to the deposition of mutated transthyretin (TTR) in various organs, commonly involving the peripheral nerves and the heart. Circulating TTR tetramers are unstable due to the presence of mutated TTR and dissociate into monomers, which misfold and form amyloid fibrils. Although there are more than 140 mutations in the TTR gene, the p.Val50Met mutation is by far the commonest. In the typical, early-onset cases, it presents with a small sensory fibre and autonomic, length-dependent, axonal neuropathy, while in late-onset cases, it presents with a length-dependent sensorimotor axonal neuropathy involving all fibre sizes. Treatment is now available and includes TTR stabilizers, TTR amyloid removal as well as gene silencing, while gene editing therapies are on the way. Its timely diagnosis is of paramount importance for a better prognosis.

1. INTRODUCTION

Variant Transthyretin Amyloidosis (ATTRv) disease is an adult-onset, autosomal, dominant, multisystemic disease due to the deposition of mutated transthyretin (TTR) in various organs [1]. Peripheral nerves and the heart are most commonly involved but the clinical phenotype is variable. There are four exons in the TTR gene, which is located on chromosome 18 [2]. TTR circulates as a homotetramer in plasma and each monomer consists of a 127-amino acid peptide with a thermodynamic predisposition to amyloid formation due to its predominant β-pleated sheet secondary structure. TTR is primarily synthesized by the liver (>90%) but other sites include the retinal pigment epithelium and the choroid plexus. In circulation, the primary functions of TTR are to carry vitamin A (in association with the retinol-binding protein) and about 15% of total thyroxine [3]. The name trans-thy-retin (transfer-thyroxine-retinol) is derived from its function in plasma. However, several important physiological roles for TTR are increasingly recognized, both in the peripheral and central nervous systems, which need to be considered when long-term gene silencing and gene editing therapies are used [4].

To date, there are more than 140 TTR mutations associated with ATTRv disease, with the most common being the p.Val50Met mutation that causes predominantly a neuropathic phenotype. In the endemic areas, the p.Val50Met mutation tends to present with early onset (age of onset before 50 years) small sensory fibre and autonomic neuropathy. In non-endemic areas, late-onset patients (age of onset after 50 years) are more common, and the phenotype is often mixed at onset and includes a non-selective sensorimotor axonal neuropathy and cardiomyopathy [5]. The clinical phenotype of ATTRv disease depends on the predominant organs affected, which in turn are determined, via poorly understood mechanisms, by the specific mutation. Disease penetrance may vary in different populations and is probably modulated by genetic and epigenetic factors [6].

The current paper will review current drug and gene therapies but important background information on ATTRv disease will also be covered to provide a more holistic view of the disease.

2. EPIDEMIOLOGY

Hereditary transthyretin amyloid neuropathy is found throughout the world with major endemic foci in Northern Portugal, Sweden and Japan [7]. More recently, smaller endemic regions have been identified in the Mediterranean, in the Balearic Islands, Cyprus and Crete [6, 8-10]. In most of Europe, TTR gene mutations are heterogeneous, while in smaller endemic foci, such as Cyprus, the most common pathogenic mutation, which is the p.Val50Met, is the only one found. Major endemic foci have both early and late-onset p.Val50Met patients [11].

3. PATHOGENESIS

Transthyretin is secreted from the liver into plasma as a homotetramer. The circulating TTR tetramer naturally dissociates in plasma into monomers, and the monomers, due to their intrinsic secondary structure, tend to misfold and polymerise into prefibrillar structures and amyloid (Fig. 1) [12].

Fig. (1).

Mutated TTR increases destabilization of the homotetramer resulting in increased levels of monomers, which misfold and aggregate via various intermediates into amyloid in various tissues beds (Reprinted from Patisiran, an RNAi therapeutic for the treatment of hereditary transthyretin-mediated amyloidosis. Neurodeger. Dis. Manag., 2019, 9(1): 5-23 with kind permission of Future Medicine Ltd.).

The phenomenon of dissociation and misfolding of TTR occurs normally in the absence of any mutation, as is seen in late-onset ATTRw (wild-type amyloidosis) cardiomyopathy, but it is enhanced by the vast majority of mutations [13]. Presumably, in late-onset ATTRw cardiomyopathy, the normal cardiac bed protein homeostatic mechanisms that normally eliminate prefibrillar/fibrillar ATTRw can not keep up. The rate of TTR homotetramer dissociation in plasma is the key rate-limiting step in amyloid formation.

The mechanisms determining phenotype, according to specific mutations of TTR, are multifactorial and poorly understood. They likely involve the amyloidogenic potential of the mutated TTR versus local tissue protein homeostatis including the impact of locally made and remotely produced chaparone molecules [14, 15]. The phenomena of variable penetrance and anticipation (which is not based on a repeat nucleotide mechanism) of the p.Val50Met mutation suggest that the genetic background of the various populations as well as epigenetic factors may play a role. More specifically, in Cypriot and Portuguese patients, complement C1Q polymorphisms modulate the age of disease onset of the p.Val50Met mutation [6, 16, 17]. Research on transgenic mice supports the role of complement in ATTR amyloidogenesis [18].

4. CLINICAL PHENOTYPE

Variant Transthyretin Amyloidosis (ATTRv) neuropathy worldwide is most commonly due to the p.Val50Met mutation and presents initially with small sensory fibre and autonomic neuropathy and subsequently involves large sensory and motor nerve fibres. Historically, called Familial Amyloid Neuropathy (FAP), it was originally described in Portugal by Andrade in 1952 [19]. Patients with early onset FAP, defined as onset before the age of 50 years, usually present with typical small sensory fibre and autonomic neuropathy phenotype. Late-onset patients, defined as onset after the age of 50 years, have both large- and small-diameter nerve fibers that also include cardiomyopathy at presentation [1]. Late-onset cases from non-endemic areas are characterized by all modalities of sensory deficits resulting from the involvement of both large- and small-diameter nerve fibers [20]. Moreover, patients with late-onset cases from non-endemic areas tend to be initially diagnosed as having chronic inflammatory demyelinating polyneuropathy [21].

Age of onset in ATTRv neuropathy is variable and anticipation is common for the p.Val50Met mutation. In both p.Val50Met and non-p.Val50Met mutations, late-onset cases may have a more aggressive disease. The typical phenotype of early-onset p.Val50Met neuropathy presents with sharp shooting pains in the feet, nausea and early satiety, diarrhoea and/or constipation, weight loss, bladder dysfunction, impotence, and changes in sweating [1]. On examination, there is dissociated sensory loss in the feet, affecting temperature and pinprick modalities, while postural hypotension, a feature of dysautonomia, is common. Occasionally, in the first few months of symptom onset, clinical examination may not be unequivocally abnormal, in which case quantitative sensory testing and/or Sudoscan testing may be employed for small diameter and autonomic fibre assessment [22]. Temperature and pain appreciation are more affected than touch and joint position sense, while later on, muscle wasting and weakness set in (Figs. 2a, and 2b).

Fig. (2).

(a) Patient diagnosed three years after onset. Dissociated sensory loss (sensory loss to temperature in blue, loss to pain in green and loss to touch in red) and no significant muscle wasting. Postural hypotension is present. Sural nerve biopsy shows severe loss of small myelinated fibres, and to a lesser degree, large myelinated fibres. Years correspond to the time from symptom onset to diagnosis. (b) Patient diagnosed eight years after onset. Dissociated sensory loss (sensory loss to temperature in blue, loss to pain in green and loss to touch in red), extending almost throughout the body, severe muscle wasting in the limbs, and catheter due to neurogenic bladder. Systolic pressure was unrecordable on standing up. Sural nerve biopsy (semithin section) shows severely depleted nerve fibres of all sizes and replaced by amorphous material consisting of amyloid. Years correspond to the time from symptom onset to diagnosis.

In late-onset cases, examination reveals all-modalities sensory deficits resulting from the involvement of both large- and small-diameter nerve fibers and early motor involvement.

A careful family history is paramount and should also include enquiry for less typical symptoms, such as carpal tunnel syndrome, lumbar canal stenosis, and cardiomyopathy both in the patient and in family members.

Following the introduction of orthotopic liver transplantation for p.Val50Met neuropathy, patient survival has increased significantly longer than the average of 10 years in the pre-transplantation era. As a result, further manifestations of this mutation have become clinically apparent. These include eye disease and central nervous system (CNS) complications [6, 23-27]. Amyloid deposition continues at these sites due to the continued production of mutated transthyretin by retinal pigment epithelium and ciliary body in the eye and the choroid plexus in the brain, respectively. In the Cypriot cohort of transplanted patients, the prevalence rate of ocular, cardiac, and central nervous complications is 60%, 20%, and 16%, respectively [6].

Ocular complications include pupillary abnormalities, kerato-conjunctivitis sicca, vitreous opacities, and glaucoma [24]. The most common CNS complications, which occur after a mean of 16 years from disease onset, are transient focal neurological episodes (TFNE), such as expressive dysphasia, dysarthria, facial distortion, and unilateral limb numbness. The TFNEs tend to be stereotyped and tend to be longer than transient ischaemic attacks. In some patients, different hemispheres are involved. The time from onset to peak in TFNEs is usually several minutes which is dissimilar to convetional transient ischaemic attacks in which onset to peak is several seconds [23]. In the Cypriot cohort of patients with CNS complications, there was 30% mortality due to cerebral haemorrhage (Fig. 3).

Fig. (3).

Gradient echo (GRE) sequence MRI demonstrating microhemorhages (small arrows) and a major hemorrhage (big arrow) in the centre of the cerebellum. Reprinted from The frequency of central nervous system complications in the Cypriot cohort of ATTRV30M neuropathy transplanted patients. Neurol. Sci., 2020, 41: 1163-1170 with kind permission of Springer nature.

The TTR mutation mainly determines the phenotype but the population’s genetic background and epigenetic factors probably modulate the phenotype (Table 1) [28].

Table 1.

Clinical manifestations and epidemiology of the most common hATTR mutations.

Mutation	Epidemiology	Peripheral Neuropathy	Autonomic Neuropathy	Cardiomyopathy	Ocular Involvement	Gastrointestinal Involvement	Renal Involvement
Val30Met (early onset)	Portugal, Brazil	++	+++	±	+	++	+
Val30Met (late onset)	Japan, Sweden, USA, Italy, France	+++	+	++	+	+	±
Val122Ile	USA	±	±	+++	±	±	±
Thr60Ala	UK, USA	+	+	+++	±	±	±
Glu89Gln	Italy, Bulgaria	++	++	++	±	+	±
Ser50Arg	Japan, France, Italy, USA	+++	+++	±	±	+	±
Phe64Leu	USA, Italy	++	++	++	±	+	±
Ile68Leu	German, Italy	±	±	+++	±	±	±
Ser77Tyr	USA, France, Israel	++	++	++	±	+	+
Ile107Val	USA, France, Brazil	++	++	++	±	±	±
Asp38Ala	Japan	++	++	++	±	±	±

Notes: The number of the “+” provides an indication of the likelihood of presence of symptoms, with “±” indicating as unknown likelihood as the symptom is present in some patients and not in others. Reprinted From Therapeutics and clinical risk management, 2020 16, 109-123 ‘Originally published by and used with permission from Dove Medical Press Ltd.). Table 1, Genotype is the main determinant of phenotype (From Diagnosis and Treatment of Hereditary Transthyretin Amyloidosis (hATTR) Polyneuropathy: Current Perspectives on Improving Patient Care, with kind permission of Dovepress).

Cardiac amyloidosis is not prominent in early-onset p.Val50Met mutation although cardiac conduction block can occur and patients are usually inserted a pacemaker prior to liver transplantation. As p.Val50Met neuropathy patients live longer nowadays, cardiomyopathy is becoming more clinically relevant. Late-onset p.Val50Met neuropathy is more often accompanied by cardiomyopathy. The latter is an infiltrative restrictive cardiomyopathy characterized by heart failure with preserved ejection fraction, a speckled appearance on ECHO and apical sparing [13]. Renal involvement is not an early feature of the p.Val50Met mutation but may manifest in the later stages of the disease [29].

5. DIAGNOSIS

In endemic regions, where family history is often present, diagnosis is usually straightforward following clinical examination and DNA testing. Furthermore in endemic regions, there are amyloid clinics where gene carriers are offered regular follow up and diagnosis is often made in less than a year from symptom onset [30]. The situation is more challenging in non-endemic areas, late-onset cases, and if there is uninformative family history and less typical symptoms. With the p.Val30Met mutation, late-onset cases may exhibit less involvement of the autonomic nervous system and are more likely to exhibit cardiomyopathy or less typical features, such as carpal tunnel, lumbar canal stenosis. The clinician needs to recognize the multisystemic involvement of ATTRv disease particularly in patients presenting with peripheral neuropathies of unknown origin. Biceps tendon rupture occurs in late-onset ATTRw, so it should raise the clinician’s suspicion about ATTRw amyloidosis.

In familial cases, the presence of the typical phenotype and the endemic TTR gene mutation is often adequate for diagnosis. Ideally, confirmation of amyloid deposits, in symptomatic tissue or more often from convenient biopsy sites, with the demonstration of the typical apple-green birefringence under polarized light with the Congo red stain, is desirable. Biopsies are usually obtained from abdominal fat, rectal mucosa, labial salivary glands, skin, nerve or myocardium [30]. The sensitivity of biopsy varies up to 80% and depends on local expertise but the patchy tissue deposition of amyloid should be appreciated. In endemic countries where ATTRv is often familial, family members are offered genetic testing and presymptomatic carriers are seen regularly in the clinic. Symptomatic carriers often undergo biopsy both to confirm tissue deposition of TTR and also because funding authorities may require biopsy proof to finance expensive therapies. In non-endemic countries, both genetic testing and testing for AL amyloidosis are indicated in suspect cases. Furthermore, because of the possibility of monoclonal gammopathy of unknown origin, a biopsy is often prudent in cases of neuropathy. This need arises especially in wild-type transthyretin amyloidosis if there is a coincidental serum gammopathy which may instead be the source of the amyloid. If a biopsy is negative for amyloid and the clinical suspicion is high, then TTR sequencing should be performed. In familial cases, the genetic diagnosis is generally known prior to symptom onset.

For peripheral neuropathy, nerve conduction studies (NCS) are performed although conventional NCS mainly assess myelinated nerve fibers larger than eight microns. Small fibre neuropathy can also be diagnosed with skin biopsy, which shows loss of epidermal nerve endings as well as denervation of sweat glands. The Sudoscan is increasingly used as a quick, non-invasive method that can be used repeatedly to assess sudomotor fibres in patients with symptoms suggestive of small sensory fibre and autonomic neuropathy [22].

For patients with possible ATTR cardiomyopathy, echocardiography with strain imaging looking for the speckled pattern as well as magnetic resonance imaging of the heart are used [13]. The use of scintigraphy with bone markers, such as ^99mTc-2,3-dicarboxypropane-1,1-diphosphonate (DPD), ^99mTchydroxymethylene diphosphonate (HMDP) or ^99mTcpyrophosphate (PYP), has become popular due to its non-invasiveness and good specificity in the correct clinical context [31]. In p.Val50Met ATTRv neuropathy, conduction abnormalities are an early sign, and electrocardiography and Holter monitoring are indicated. Similarly, in monitoring for incipient cardiac failure, N-terminal prohormone (NT-proBNP) is very useful and can be used serially to monitor progress [13].

6. TREATMENT

Treatment in ATTRv neuropathy currently aims at eliminating the source of mutated TTR and stabilizing the TTR tetramer so that there is no further dissociation and deposition of misfolded TTR peptides in the various organs. TTR amyloid removal is still under clinical evaluation. Since ATTRv neuropathy is a multisystemic disease, symptomatic treatment needs to be provided by a multidisciplinary team, which will not be covered in this review.

6.1. Liver Transplantation

Orthotopic liver transplantation was historically the first treatment introduced to eliminate the source of mutated TTR since the liver is the main site of TTR production (accounts for 99% of circulating TTR) and was first performed in 1990 [32]. Life survival at 20 years after liver transplantation is 55% compared to a median 10-year survival associated with the p.Val50Met mutation prior to transplantation [33]. However, the transplanted liver produces normal TTR, which continues to be deposited in previously seeded tissues, such as the heart [34]. In addition, late-onset p.Val50Met and non- p.Val50Met mutation patients do worse than early onset p.Val50Met mutation following liver transplantation. Furthermore liver transplantation does not prevent the CNS or eye complications of ATTRv neuropathy [23, 27, 35]. Liver transplantation is being abandoned following the introduction of TTR stabilizers and gene-silencing therapies.

6.2. Drug Therapies - Oral Stabilizers

Oral TTR stabilizers include Tafamidis (approved in Europe in 2011) and the non-steroidal anti-inflammatory drug Diflunisal (used off-label), both of which dock into the thyroxine carrier sites of the TTR tetramer, stabilize it, and reduce the dissociation rate of the homotetramer [36-38]. Tetramer dissociation is the rate-limiting step in TTR amyloid formation.

Several trials on Tafamidis, which include both p.Val50Met and non-p.Val50Met patients, have shown a reduction in neuropathy progression in most but not all patients [39-43]. Advanced neuropathy and/or old age are bad prognostic factors for a good response. In addition to impacting the sensorimotor aspect of ATTR neuropathy, there is also evidence, that if given early, progression of the autonomic component may also be retarded [44]. Tafamidis, both at 20 mg and 80 mg per day, has been shown to reduce mortality and cardiovascular-related admissions in patients with ATTRv and ATTRw cardiomyopathy, with the latter dose being the most effective [45, 46]. Preliminary reports suggest that Tafamidis may stabilise renal function and proteinuria, especially if used early in the course in p.Val50Met patients [47, 48]. As mentioned above, eye and CNS complications in patients with ATTRv neuropathy are becoming an increasing cause of morbidity and mortality in the current era of anti-amyloid therapies. Transthyretin is secreted in the CNS by the choroid plexus and in the eye by the retinal and ciliary body epithelia. Amyloid deposition is expected to occur along the same lines as in the peripheral nervous system and the heart. Therefore, it is perhaps important that 20 mg of Tafamidis meglumine (the approved dose for peripheral neuropathy) has been shown to penetrate both the cerebrospinal fluid and vitreous, be it at substoichiometric levels [49]. Clinical studies to demonstrate the impact of Tafamidis, at 20 mg or higher doses, on the CNS and eye complications are urgently needed since there is a large pool of liver transplanted and anti-sense oligonucleotide/siRNA treated patients currently expected to develop these complications.

Diflunisal, a non-steroidal antiflammatory drug, has also been found to be a TTR stabilizer for both native and mutated TTR [37, 38]. A phase 3 trial, using 250 mg twice a day, of diflunisal for ATTRv neuropathy has shown a significant reduction in the rate of progression of neuropathy over a 2-year period and preserved quality of life [40]. A subsequent study involving a Japanese cohort with a mean follow-up of 38 months showed that diflunisal had a sustained benefit and was well tolerated, although in 3 out of 40 patients, the drug had to be discontinued due to renal dysfunction in two patients and thrombocytopenia in one patient [50]. Although diflunisal is not licenced for ATTR cardiomyopathy, it is used off-label to that effect. A recent systematic review showed that diflunisal use was associated with decreased mortality and a reduced number of orthotopic heart transplants in ATTR cardiomyopathy [51]. As with neuropathy, a number of patients had to stop treatment due to gastrointestinal and renal side effects [51]. There are no clinical data on the penetration of diflunisal into the CNS and eye and, therefore, its impact on transthyretin amyloidosis in these sites is unknown.

A novel oral TTR stabilizer, AG10, has entered phase 3 trials for both ATTRv neuropathy and cardiomyopathy [52, 53]. Tolcapone, a drug already approved for Parkinson’s disease, has been shown to bind and stabilize leptomeningeal TTR variants in vitro and in view of the fact that it penetrates the blood-brain barrier and offers hopes in the treatment of ATTRv CNS complications [54]. A phase 2a study in normal subjects and in patients with the p.Val50Met mutation has demonstrated the ability of tolcapone to stabilize TTR [55].

6.3. Drug Therapies - TTR Deposit Removal

A combination therapy of doxycycline and tauroursodeoxycholic acid (TUDCA) has been shown, both in p.Val50Met transgenic mice and in a phase 2 study, to have potentially a beneficial effect [56, 57]. Doxycycline removes fibrillar TTR while TUDCA removes prefibrillar TTR. Monoclonal antibodies against serum amyloid P (SAP) and TTR are currently being investigated [58].

6.4. Gene Silencing Therapy

Recently, two gene silencing therapies have been approved for downregulating, by more than 80%, TTR production in the liver [59, 60].

6.5. Small Interfering RNAs

Patisiran (approved in Europe in 2018) consists of a small interfering RNA (RNAi), encapsulated in lipid nanoparticles, given intravenously every three weeks and taken up by liver hepatocytes via receptor-mediated endocytosis. The siRNAs are 21-23 nucleotide long double-stranded RNA molecules that target and cleave mRNA after their incorporation into the RNA-induced silencing complex (RISC). Patisiran mediates the degradation of TTR mRNA in the cytoplasm by targeting the 3’ untranslated region of TTR mRNA. Following intravenous infusion, the lipid nanoparticle-encapsulated Patisiran siRNAs are opsonised by apolipoprotein E and enter the hepatocytes by means of the ApoE-binding cell surface receptor [12]. Preclinical and phase 2 studies have shown a dose-dependent reduction in TTR in both normal controls and in patients with TTR amyloidosis [61-63]. In animal studies, there was a regression of existing TTR deposits [63]. The APOLLO trial was a phase 3 double-blind trial of patisiran, given at 0.3 mg/kg every three weeks, lasting 18 months, and included patients with p.Val50Met mutation of both early and late-onset as well as non-p.Val50Met mutations [59]. The patisiran-treated patients had a significant improvement in their mNIS+7 score (modified Neuropathy Impairment Score), a score that combines both clinical and neurophysiological neuropathy-related measurements, compared to worsening in the placebo-treated patients [59]. Quality of life as well as several secondary outcomes also improved in the treated but worsened in the untreated group. A 12-month follow-up study to APOLLO has confirmed both the safety and effectiveness of patisiran [64]. A sub-study of APOLLO, examining the effect of patisiran on cardiomyopathy, showed a halt or even a reversal in the progression of cardiomyopathy [65]. There are anecdotal reports and a phase 3b trial of the use of patisiran in patients with progressive ATTR neuropathy following liver transplantation with very encouraging results [66, 67]. Although there are no direct comparison studies, there is some preliminary evidence that patisiran may be more effective than tafamidis in ATTRv neuropathy [68]. Animal experiments show that systemically administered TTR siRNA had no effect on cerebrospinal TTR levels which may be a mixed blessing in that any neuroprotective effect of TTR on the brain is spared, but on the other hand, an effect on CNS amyloidogenesis may be unlikely [69].

Vutrisiran (ALN-TTRSC02) is a second-generation siRNA, a single dose of 25mg of which, in a phase 1 study, resulted in a maximum 80% TTR reduction for 90 days. It is estimated that 25 mg SC of vutrisiran every three months will result in the same suppression of TTR as intravenous patisiran thrice weekly [70]. A phase 3 trial, Helios A, is currently underway comparing the two siRNAs. Similarly, a ligand conjugated antisense oligonucleotide ION-682884-CS3, related to inotersen, is being tested in a phase 3 study (NEURO-TTRansform Study) and is given 4 weekly and compared to 300 mg sc weekly of inotersen [71].

6.6. Antisense Oligonucleotides (ASOs)

Inotersen (approved in Europe in 2018) is an antisense oligonucleotide administered subcutaneously. Antisense oligonucleotides are single stranded 20 nucleotides that bind to complementary mRNA in the nucleus and destroy it via the RNase H1 mechanism [72]. Preclinical studies in a transgenic mouse model carrying the human TTR Ile84Ser mutation showed that TTR ASOs suppressed hepatic TTR mRNA and serum TTR by 80% [73]. The Neuro-TTR trial, a 15-month-long phase 3 trial of inotersen, an ASO that binds the 3’ untranslated part of the human TTR RNA, involved administering inotersen subcutaneously three times a week in the first week and then once weekly [60]. The treatment group fared better than the control group on the mNIS+7, a comprehensive scale of neuropathy, as well as several quality-of-life assessments. There were two serious adverse events in the treatment group, which were glomerulonephritis and thrombocytopenia, occurring in 3% of patients each. The long-term efficacy and safety of inotersen were assessed in an open-label two-year extension study, demonstrating slowing of disease progression and reduced deterioration of quality of life [71]. No extra safety issues were observed. Currently, a phase 3 study on ATTR neuropathy with a ligand-conjugated ASO to facilitate hepatic cell receptor uptake and improve potency and safety is underway [74]. As with patisiran, there is evidence that inotersen is safe and effective in stabilizing hereditary ATTR cardiomyopathy [75]. Regarding the CNS complications of ATTRv neuropathy, there are animal data that systemic administration of ASOs does not impact choroid plexus TTR production but intrathecal delivery does [76]. Thus, it is likely that systemic delivery of inotersen will not be effective in preventing CNS or eye complications [77]. Inotersen has been anecdotally used in patients following orthotopic liver transplantation with some success [78].

6.7. Gene Editing

Patients on three weekly patisiran dose are subjected to regular intravenous steroids while those on weekly subcutaneous inotersen administration are exposed to potentially serious side effects. Thus, lifelong gene silencing using these two molecules or even their next-generation subcutaneous successors is not without risks. Recently, an alternative to mRNA targeting-based gene silencing has been tested preclinically using the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system to achieve in vivo knockout of the transthyretin gene [79]. A single injection resulted in a 97% reduction of serum TTR for one year. In a phase 1 trial, NTLA-2001, a lipid nanoparticle delivery system carrying a single guide RNA targeting the human TTR and the mRNA of Streptococcus pyogenes, CAS9 protein was injected once in six patients with ATTR amyloidosis [80]. Serum TTR was found to be reduced by 87% by day 28 with the higher dose used; however, long-term data are eagerly awaited.

CONCLUSION

ATTR amyloidosis is an excellent example of the triumph of translational medicine in neurology, whereby progress in molecular medicine actually translates into saving lives. ATTRv neuropathy, a worldwide lethal autosomal dominant multisystemic disease, is now treatable by a variety of approaches that include TTR stabilizers, TTR amyloid removal, and gene silencing either via RNA editing or, in the near future, TTR gene editing. Wildtype ATTR cardiomyopathy, a much commoner condition, is similarly treatable with TTR stabilizers and perhaps in the future with gene silencers when phase 3 studies become available. There are,however, several unmet needs that need to be met and these include eye and CNS complications. These manifestations are becoming an increasing cause of morbidity and mortality since the above therapies do not impact these privileged tissue sites. Perhaps other modes of delivery may be needed but there is certainly scope for optimism.

LIST OF ABBREVIATIONS

ATTRv

Variant Transthyretin Amyloidosis

ATTRw

Wild Type Amyloidosis

CNS

Central Nervous System

CRISPR-Cas9

Clustered Regularly Interspaced Short Palindromic Repeats and Associated Cas9 Endonuclease

DPD

^99mTc-2,3-Dicarboxypropane-1,1-diphosphonate

FAP

Familial Amyloid Neuropathy

HMDP

^99mTchydroxymethylene Diphosphonate

NT-proBNP

N-Terminal Prohormone

PYP

^99mTcpyrophosphate

RISC

RNA-Induced Silencing Complex

RNAi

Small Interfering RNA

TFNE

Transient Focal Neurological Episode

TTR

Transthyretin

TUDCA

Tauroursodeoxycholic Acid

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.