First in man in vivo delivery of CRISPR-Cas9 targeting Transthyretin Amyloidosis

Authors: Dr Christos Georgiadis, October 2021.

While this year has to most felt as though life came to a standstill, the field of genome editing has experienced unprecedented momentum. With CRISPR basking in the limelight following on from the joint Emmanuelle Charpentier and Jennifer A. Doudna 2020 Nobel Prize there has been a phenomenal growth in R&D and industry partners creatively exploring the platform’s capabilities.

It is hard to believe that a genome editing technology brought to light a mere 9 years ago would make it through the scrutiny and stringency of regulatory authorities and into patients, which undoubtedly highlights the power and accessibility of the technology and the unmet clinical need that it can cater for.

We have seen CRISPR used in a number of ex vivo applications where genome editing is performed on patients’ or healthy donor cells outside the body and the modified cells are re-infused back into the patient. Notable have been medicinal products developed by CRISPR therapeutics investigating autologous, gene-edited hematopoietic stem cell (HSCs) therapy for patients suffering from severe hemoglobinopathies (CTX001; NCT03745287), where CRISPR-Cas9 is employed to modify a patient's HSCs so that they produce high levels of foetal haemoglobin and re-infused back into the patient in the form of a stem cell transplant, and separately, ‘off-the-shelf’ CRISPR/Cas9 gene-edited CAR T cells for the treatment of CD19+ malignancies (CTX110; NCT04035434).

A phase I study assessing safety and efficacy of CRISPR/Cas9-edited ‘universal’ CAR T cells in paediatric B cell acute lymphoblastic leukaemia (B-ALL) is also currently underway in the UK at the Great Ormond Street Institute of Child Health (NCT04557436).

Delivery of genome editing reagents directly to the patient, known as in vivo delivery, holds great promise for the treatment of systemic disease, however poses a significant challenge with regards to delivery method, biodistribution, efficacy and off-target activity and hence comes with greater risks.

The study conducted by Intellia in partnership with Regeneron constitutes the first in vivo delivery of CRISPR genome editing reagents in humans.

The study reported in the NEJM set out to demonstrate safety and efficacy of CRISPR-Cas9 editing of transthyretin in patients with hereditary amyloidosis with polyneuropathy (ATTRv-PN; v for “variant”) (Gillmore, Maitland et al. 2021). ATTR is a rare debilitating and if untreated often fatal systemic disease, characterised by an abnormal build-up of amyloid deposits of aggregates of misfolded transytheritin proteins in organs and tissue leading to progressive polyneuropathy and cardiomyopathy.

ATTR-PN has an autosomal dominant mode of transmission and is therefore hereditary affecting 1.1 in 100,000 people with an onset of as early as 30 years of age and with a prognosis of 3-15 years. Over 100 different mutations in the TTR gene have been linked to ATTR-PN.

Current treatment options operate by either reducing amyloid formation through stabilisation of the tetrameric form of TTR or by entirely inhibiting TTR protein formation by targeting its mRNA, with the latter associated with improved neuropathic endpoints. While a level of functional improvement and symptom relief can be achieved, the nature of the therapeutics necessitates their long-term administration which in turn prolongs exposure to the treatment agents exacerbating side effects.

ATTR being a monogenic disease therefore presents an ideal target for the CRISPR/Cas9 platform ensuring durability of treatment following single dose administration. Moreover, TTR is associated with restricted function in thyroxine and vitamin A transport suggesting that its CRISPR-mediated knockdown is unlikely to result in adverse physiological effects and can be managed through vitamin A supplementation.

Intellia Therapeutics and Regeneron Pharmaceuticals developed a proprietary therapeutic, NTLA-2001 (NCT04601051), based on lipid nanoparticle (LNP) technology designed to favour targeting of hepatocytes, known as liver tropism, which is used as a vehicle for the transport of ribonucleoprotein complex (RNP) of Streptococcus pyogenes Cas9 protein and a single guide RNA (sgRNA) targeting the endogenous TTR gene. Once the CRISPR/Cas9-sgRNA complex identifies its target sequence it creates a double-stranded DNA break which will undergo repair by the cellular non-homologous end joining pathway (NHEJ). Relying on the error-prone nature of NHEJ, insertions/deletions (indels) will be introduced at the site disrupting the coding sequence and hence ablating protein expression.

Preclinical studies of NTLA-2001 in transgenic mice revealed impressive genome editing efficacies with sustained suppression of serum TTR protein levels at 12 months. Similar observations were reported in cynomolgus monkey studies where infused LNPs demonstrated rapid distribution to the liver and achieved near complete >94% reduction of serum TTR over 12 months with no associated adverse effects.

Across 6 patients with ATTR-PN amyloidosis, 3 receiving a single intravenous 0.3 mg/kg dose of NTLA-2001 achieved impressive reductions in serum TTR protein levels by ≈87% and in one patient as high as 96% while 3 patients dosed with 0.1 mg/kg dose achieved 52% mean reduction by day 28. Alternative antisense oligonucleotide or RNAi therapies against ATTR can typically lead to a reduction of up to 80%, however maintenance of this reduction relies on serial and chronic administration of the agents.

While monitoring is ongoing to determine the long-term safety and efficacy of the treatment and dose escalation studies in a larger number of patients is currently in the enrolment phase, this study represents a milestone in genome editing bringing the ‘new kid on the block’, CRISPR/Cas9, to the forefront of therapeutic development. NTLA-2001 will undoubtedly be the first of many CRISPR-based therapeutics to follow suit with Intellia and Regeneron already developing pipelines for haemophilia A and haemophilia B amongst others.


Gillmore, J. D., M. L. Maitland and D. Lebwohl (2021). "CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. Reply." N Engl J Med 385(18): 1722-1723.