CRISPR-Cas9: Gene Editing for hATTR Amyloidosis

CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

Gillmore JD, Gane E, Taubel J, et al.

N Engl J Med. 2021 Aug 5;385(6):493-502. [Full text]

NEJM Video Summary

Summary by Shabnam Elahi

Transthyretin (ATTR) amyloidosis is a fatal disease characterized by deposition of amyloid fibrils composed of transthyretin (TTR) in tissues, predominantly in nerves and the heart. It results in progressive polyneuropathy and/or cardiomyopathy with subsequent heart failure and can be acquired (wild-type) or hereditary. Hereditary ATTR amyloidosis (hATTR) affects approximately 50,000 individuals worldwide, and is a result of one of 100 autosomal dominant mutations identified in the TTR protein [1]. Mortality in ATTR amyloid cardiomyopathy occur approximately 2 to 6 years after diagnosis and 4 to 17 years in patients with amyloid polyneuropathy without cardiomyopathy [2].

Given the high morbidity and mortality associated with hATTR amyloidosis, there have been several treatments developed aiming to improve symptoms and prolong survival. Current therapies act by reducing amyloid formation through stabilization of the tetrameric form of TTR (diflunisal or tafamidis), or by inhibiting TTR protein synthesis by degradation of TTR mRNA (inotersen or patisiran). Liver transplantation has also become an available therapy as TTR is primarily produced by hepatocytes.

Current therapies are primarily limited by the requirement for long term administration and patients receiving TTR-stabilizing agents (diflunisal or tafamidis) still experience disease progression. Inotersen is associated with adverse effects including glomerulonephritis and decreased platelets [3]. In contrast, the CRISPR-Cas9–based in vivo gene-editing therapy theoretically only requires a single infusion to permanently silence TTR production. This phase 1 study explores the safety of gene editing as a strategy to provide a more sustained TTR reduction, and perhaps one day improve clinical outcomes for patients with hATTR amyloidosis.

Patient population and Design

This study design was an open-label, phase 1 clinical trial evaluating the safety and pharmacodynamic effects of NTLA-2001 (CRISPR-Cas9–based in vivo gene-editing therapy) targeting TTR in human hepatocytes in adults with hATTR with polyneuropathy with or without cardiomyopathy.

Six patients were enrolled in the study from centers in Auckland, New Zealand and London, UK. Eligibile patients were 18 -80 years old and lacked of access to approved treatments for ATTR amyloidosis. Excluded patients were those with non-ATTR amyloidosis, known leptomeningeal ATTR amyloidosis, or a history of receipt of RNA-silencing therapy. Of note, prior use of TTR stabilizers was permitted with a washout period (3 days for diflunisal).

Each patient received a single intravenous infusion of NTLA-2001 at a dose of either 0.1 or 0.3 mg/kg of body weight. Prior to drug administration patients were pre-treated with oral dexamethasone, IV steroids, and an H1 and H2 blocker. Serum samples were obtained at baseline, 7, 14, and 28 days for measurement of TTR protein levels with a validated enzyme-linked immunosorbent assay.


The study investigated a primary outcome and safety outcome. The primary outcome was to determine pharmacodynamic effects of NTLA-2001 by measuring concentrations of TTR in serum at baseline and at days 7, 14, and 28 days. The 28 day mark was selected as this was the time at which the drug effect had reached its permanent nadir in preclinical studies.  The safety assessments throughout the study period included monitoring for: adverse events, lab abnormalities (hematology, chemistries, liver enzymes, coagulation parameters, urinalysis), vitamin A and retinol binding protein levels, thyroid function, inflammatory and immune markers, physical exam, and ECG.   


The six patients included four males and two females and carried three different hTTR mutations. Three patients had received no previous therapy, and the remaining three had previously received diflunisal. All participants had sensory polyneuropathy and NYHA HF class of I.

Regarding the primary outcome, investigators observed a reduction in serum TTR protein concentration from baseline by day 14 after NTLA-2001 administration to day 28. By day 28, the dose of 0.1 mg/kg group had a mean TTR reductions of 52% (with a range of 47 to 56%) and the 0.3 mg/kg dose group had a mean TTR reduction of 87% (with a range of 80 to 96%). Based on these results, investigators concluded NTLA-2001 effect on TTR reduction to be dose-dependent, and reproducible.

Regarding safety outcomes, NTLA-2001 treatment was associated with adverse events of only mild severity with one reported infusion-related reaction. No serious adverse events were observed. The authors noted no significant laboratory abnormalities.

Of note, data were collected by study investigators in New Zealand and the United Kingdom and were analyzed by the sponsors.


CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis is one of the first studies exploring gene editing as a therapeutic strategy for treatment of hATTR amyloidosis, a disease with significant morbidity and mortality with limited therapeutic options. The investigators observed a TTR reduction from NTLA-2001 that was dose-dependent and reproducible.

While this is a promising therapeutic, as a phase 1 study, which included a very small number of patients with limited follow-up, only a dose effect with relative safety can be surmised. The descriptive analysis of results was limited to comparison of mean reduction in TTR concentration from baseline in the low dose and high dose groups, without statistical analysis. The results correlate with prior findings from preclinical studies, but phase 2 safety then phase 3 efficacy studies will still need to be completed to impact clinical practice.

Specifically phase 2 should look at longer term safety and adverse effects. Particularly of interest are any effects of possible off-target gene editing. While, gene editing tools aim to target a specific gene, they can edit non-target genes as well. Preclinical studies investigating off-target editing determined that the DNA structural variants induced by CRISPR-Cas9 were related to the end-joining at the TTR on-target site and were predicted to be of low risk.

Though still early in the discovery process, CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis, presents early results that provide a proof of concept that CRISPR-Cas9–mediated gene editing has potential to be a therapeutic option for treatment of hATTR.

F: Follow up28 days
R: RandomizationNo
I: Intention to treatNo, proof of concept study
S: Similar at baselineYes
B: BlindingNo
E: Equal treatmentnot discussed
S: Source (funding)Intellia Therapeutics and Regeneron Pharmaceuticals

  1. Hawkins PN, Ando Y, Dispenzeri A, Gonzalez-Duarte A, Adams D, Suhr OB. Evolving landscape in the management of transthyretin amyloidosis. Ann Med 2015; 47:625-38.
  2. Merlini G, Coelho T, Waddington Cruz M, Li H, Stewart M, Ebede B. Evaluation of mortality during long-term treatment with tafamidis for transthyretin amyloidosis with polyneuropathy: clinical trial results up to 8.5 years. Neurol Ther 2020;9:105-15.
  3. Gertz, Morie A., et al. Inotersen for the treatment of adults with polyneuropathy caused by hereditary transthyretin-mediated amyloidosis. Expert review of clinical pharmacology. 12.8 (2019): 701-711.