In vivo gene editing works in humans: Results of a phase 1 clinical trial for TTR amyloidosis

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The recent report by Gillmore et al.¹ describes the outcome of a small phase 1 clinical study of in vivo gene editing in six patients with hereditary transthyretin (hATTR) amyloidosis using a lipid nanoparticle (LNP) encapsulating mRNA for Cas9 combined with a single short guide RNA (sgRNA) targeting the transthyretin (TTR) gene. Intravenous (i.v.) administration of this agent (NTLA-2001) was safe and resulted in substantial reductions in serum TTR protein concentrations (47% to 96%) mediated by introduction of double-strand breaks (DSBs) into the TTR gene in hepatocytes, causing indels in TTR that disrupted its expression in a dose-dependent manner. This paper is the first report of clinical safety and molecular efficacy of i.v. delivery of CRISPR-Cas9 to treat a genetic disorder. Given the broad range of genetic disorders involving the liver, these findings could set the stage for a number of future CRISPR-based clinical products. This study only involved gene disruption, rather than gene repair, and only directly addresses the feasibility of treating conditions in which knockdown of expression is the primary goal. Therapy for disorders whose treatment requires the additional editing components needed for homology-directed repair (HDR) could potentially be less efficient. Further studies are needed to determine whether such disorders could be treated using this same platform technology.

hATTR amyloidosis is an autosomal dominant and progressively fatal disease caused by more than 100 different mutations in the TTR gene. It is characterized by deposition of misfolded TTR protein over time, mainly in the autonomic and peripheral nerves, but also in the kidneys or heart, causing cardiomyopathy.^2,3 TTR is a homotetrameric serum- and CSF-circulating protein responsible for the transport of retinol and thyroxine. The primary source of the serum circulating protein is the liver, but the retinal pigment epithelium (RPE) cells in the eye and the choroid plexus in the brain are also major TTR production sites.³ When the tetramer contains at least one subunit with mutations, it becomes thermodynamically unstable and therefore prone to dissociation. The unstable monomers misfold, become prone to aggregation, and aggregate into toxic oligomers and amyloid fibers that deposit along nerve fibers.⁴ This mechanism seems to be common to any and all of the known amylodogenic TTR mutations, therefore leading to the notion that strategies aiming to stabilize the tetramer or reduce overall protein expression can be used as a treatment for hATTR amyloidosis.

Current treatment options for hATTR amyloidosis are limited and not definitive. These include liver transplantation, tetramer stabilization (with tafamidis or diflunisal) or gene expression reduction with siRNA (patisiran, siRNA-GlucNAc conjugate), or ASO (inotersen, 2′-O-methoxyethyl-modified antisense oligonucleotide), all of which show safety and efficacy (with some degree of symptom relief), halt disease progression, and prolong survival, but none with 100% efficacy.5, 6, 7, 8, 9 However, none of these approaches provide a mechanism for long-term persistence, and an unmet need still exists for a potential one-time permanent treatment for this condition.

Preclinical studies of NTLA-2001 in transgenic mice and cynomolgus monkeys demonstrated sufficient safety and biological efficacy to justify institution of a phase 1 clinical trial. Patients with hATTR amyloidosis were enrolled at sites in New Zealand and the United Kingdom. Of the six patients enrolled, three had the p.T80A mutation, two had the p.S97Y mutation, and one had the p.H110D mutation. These patients had sensory polyneuropathy and mild or no cardiac involvement (New York Heart Association class 1 status). The first two dosage groups (0.1 mg/kg and 0.3 mg/kg) reported an excellent safety profile.

Remarkably, the 0.1 mg/kg dosage group demonstrated a mean reduction in circulating TTR of 52% (range 47% to 56%), while the 0.3 mg/kg group demonstrated reduced serum TTR by 87% (range 80% to 96%). This degree of reduction by i.v. injection is likely to be clinically important for the treatment of patients with ATTR amyloidosis. The fact that this level of response was seen at the 0.3 mg/kg dose most likely suggests that this therapy could be scaled sufficiently for the therapy to be offered to most or all patients with this diagnosis.

Importantly, if this were to translate to other genetic conditions, this level of efficiency could be quite important for these other conditions as well. One such condition, alpha-1 antitrypsin liver disease, would be expected to respond to 50% or greater reduction of the mutant protein.¹⁰ While the single-edit gene knockout approach is designed only to treat toxic gain-of-function mutations, the surprisingly positive finding that the lipid nanoparticle (LNP) platforms is efficient enough to work for this condition bodes well for future therapies that require delivery of a homology-directed repair (HDR) template in addition to Cas9 and one or two sgRNAs.

However, as exciting as it is to see the huge success described in this report with a safe reduction of circulating TTR protein after liver gene editing, one cannot forget the additional sites of TTR production, such as the choroid plexus, and we cannot deem this approach as a cure for hATTR. In vivo CRISPR-Cas9 gene editing is a powerful tool, showing here that its potential is more than just a promise on paper or in small animals. But the scientific community still needs to find the way to further improve the technology and make it a reality to treat other organs.