This activity was funded by Alnylam Pharmaceuticals. The medical content was developed independently by M3 and Professor Violaine Planté-Bordeneuve.

Highlights from

Amyloidosis Conferences



A single-dose alternative to gene silencing therapies in hATTR? Results from the first in-human demonstration of CRISPR-based gene editing therapy

Take-home messages
  • Current therapies for hATTR amyloidosis require chronic, lifelong administration and do not provide 100% knockdown of TTR

  • NTLA-2001 is a novel CRISPR/Cas9 based in vivo gene editing therapy that targets the TTR gene

  • Experiments in cell cultures and an animal model have demonstrated effective knockdown of TTR with NTLA-2001 with no evidence of off-target gene editing

  • Interim results from a proof-of-concept study in human patients show promising results with low concentrations of NTLA-2001; results with higher doses are eagerly awaited

Therapeutic need in hATTR amyloidosis

Hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) is a rare, progressive and fatal disease caused by amyloid deposits made up of misfolded transthyretin (TTR) monomers, that accumulate throughout the body, particularly in the heart and nerves.

At present, the main therapeutic strategy for hATTR amyloidosis is to reduce the concentration of circulating TTR monomers, either by removing them or stabilising the precursor protein, and this has shown some success; gene silencing therapies have been shown to reduce serum TTR by an average of 80%, providing benefits in neuropathy. However, these require lifelong administration and do not achieve 100% knockdown of TTR, leaving room for improvement.

Evidence from other types of amyloidosis shows that greater TTR knockdowns result in improved outcomes:

  • In AL amyloidosis outcomes are better in patients who achieve complete response (100% knockdown) versus patients who achieve very good partial response (90% knockdown)
  • In AA amyloidosis, relative risk of death is 4x higher in patients with median annual serum amyloid A (SAA) protein concentration of 4-9 mg/L versus patients with normal SAA protein concentration (<4 mg/L) concentration

Emerging evidence suggests that the same is true for ATTR amyloidosis, highlighting a need for therapies that provide greater reductions in serum TTR.

NTLA-2001: mechanism of action

At the 3rd European ATTR meeting, Professor Julian Gillmore, Head of the Centre for Amyloidosis and Acute Phase Proteins, UCL, presented data for a novel CRISPR-based in vivo gene editing therapy NTLA-2001, designed to reduce expression of TTR.

NTLA-2001 consists of a lipid nanoparticle (LNP) containing Cas9 mRNA and a single guide RNA (sgRNA) molecule specific to the TTR gene. When administered by intravenous (IV) infusion, the LNP is transported to the liver where it is taken up by LDR receptors on hepatocytes. In the cytoplasm of the hepatocyte, the Cas9 mRNA is translated, producing the functional Cas9 enzyme which interacts with the sgRNA to form a CRISPR ribonucleoprotein (RNP). The CRISPR RNP is transported into the nucleus, where it induces unwinding of the chromosomal DNA, binds to the TTR gene and induces DNA cleavage. Endogenous DNA repair of the breaks introduce insertions or deletions of bases, resulting in frameshift mutations that reduce the production of functional TTR protein.

NTLA-2001: ex vivo and animal studies

Professor Gillmore presented results from experiments in cultured primary human hepatocytes, that showed NTLA-2001 had an EC90 (the concentration where a 90% reduction in TTR protein production is routinely seen) of 0.574 nM. Above this concentration, NTLA-2001 demonstrated saturating levels of gene editing, accompanied by >95% reduction of TTR serum concentrations.

Despite these promising data, NTLA-2001 was not ready to be brought to the clinic; Professor Gillmore’s team next determined if there was as any off-target gene editing occurring when NTLA-2001 was used at a therapeutic concentration. Genome-wide screening (using computational and laboratory techniques) detected seven potential off-target binding sites for NTLA-2001, but all of these were in non-coding regions of DNA, suggesting they would be unlikely to have deleterious effects. Experiments comparing the rate of on-target versus off-target editing with NTLA-2001 found no detectable off-target editing with pharmacologic concentrations of sgRNA (up to 3-fold greater than the EC90).

Preclinical in vivo studies in monkeys, using surrogate LNPs with sgRNA specific to the monkey TTR gene, measured the mean reduction from baseline in serum TTR in monkeys receiving NTLA-2001. Doses of 1.5, 3 or 6 mg/kg were used, with three animals per dose. A single dose at 3 mg/kg demonstrated a durable, >95% reduction in TTR at Day 28. Safety studies identified a dose range in which no adverse events (AEs) were observed.

NTLA-2001: in-human study

A two-part, open-label, phase I study of NTLA-2001 is now underway in adults with hATTR with polyneuropathy. Part one uses a single-ascending dose methodology, in which a minimum of three patients per dose group will receive NTLA-2001 by IV infusion, starting with a dose of one-tenth of the allometrically scaled dose at which no AEs were identified in the monkey studies. The primary objectives are to evaluate safety and tolerability, measure the pharmacokinetics and pharmacodynamics, and assess the reduction in TTR serum concentration.

At the EU-ATTR meeting, Professor Gillmore presented interim data from the first six patients to receive NTLA‑2001 (three patients receiving 0.1 mg/kg and three patients receiving 0.3 mg/kg). NTLA-2001 was generally well-tolerated in the acute phase and no serious AEs were reported. All reported AEs were grade 1 and mainly unrelated to treatment; one patient experienced a mild infusion-related reaction which did not affect their participation. No liver toxicity or coagulopathy was observed.

A dose-dependent reduction in serum TTR concentrations were observed by Day 7 and continued through to Day 28. At Day 28 the mean (range) reductions in TTR concentration from baseline were:

  • 0.1 mg/kg group - 52% (47%-56%)
  • 0.3 mg/kg group - 87% (80%-96%)

A knockdown of 87%, as shown in the 0.3 mg/kg group, would be expected to produce a positive outcome in terms of neuropathy.


This first demonstration of CRISPR-based in vivo gene editing in humans has shown that systemic administration of NTLA-2001 in patients with hATTR amyloidosis and polyneuropathy causes a profound and dose-dependent reduction in serum TTR protein concentration, providing proof-of-concept for a promising new therapeutic strategy.

Part two of the study presented by Professor Gillmore will see NTLA-2001 given to an expanded cohort of patients, but first the dose will be further escalated in six more patients to identify the optimum dose. Professor Gillmore hopes that this study will lead to improved outcomes for patients:

"…further dose escalation is ongoing in this first in-human study, with the aim of achieving greater reduction in TTR than that provided by currently available agents, which is expected to translate into improved clinical benefits for patients - in other words, improvement in autonomic symptoms, better mobility and fewer cardiac hospitalisations for heart failure".

AA: amyloid A; AL: amyloid light chain; CRISPR: clustered regularly interspaced short palindromic repeats; NYHA: New York Health Association; UCL: University College London

Based on: Gillmore J D, Taubel J et al. In vivo CRISPR/Cas 9 editing of the TTR gene by NTLA-2001 in patients with transthyretin amyloidosis. Presented at the 3rd European ATTR amyloidosis meeting for patients and doctors, 6-8 September 2021.

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