Patient-Specific In Vivo Gene Editing to Treat a Rare Genetic Disease

Abstract

Base editors can correct disease-causing genetic variants. After a neonate had received a diagnosis of severe carbamoyl-phosphate synthetase 1 deficiency, a disease with an estimated 50% mortality in early infancy, we immediately began to develop a customized lipid nanoparticle-delivered base-editing therapy. After regulatory approval had been obtained for the therapy, the patient received two infusions at approximately 7 and 8 months of age. In the 7 weeks after the initial infusion, the patient was able to receive an increased amount of dietary protein and a reduced dose of a nitrogen-scavenger medication to half the starting dose, without unacceptable adverse events and despite viral illnesses. No serious adverse events occurred. Longer follow-up is warranted to assess safety and efficacy. (Funded by the National Institutes of Health and others.).

Figure 1.

Timeline from Birth to Second Treatment with K-abe.

Figure 2.. Preclinical Studies.

Panel A shows how a single-strand DNA oligonucleotide cassette harboring the CPS1 Q335X variant sequence and three other variants was inserted into the endogenous mouse Rosa26 locus in mouse zygotes using CRISPR-Cas9 to introduce a double-strand break in the Rosa26 locus, followed by homology-directed repair with the cassette. Panel B shows whole-liver corrective adenine base editing of the CPS1 Q335X variant in Rosa26-Q335X mice. Following a single treatment with the toxicology batch of k-abe at the indicated dose, each mouse had multiple liver samples, distributed throughout the liver, collected upon necropsy (N = 8 samples per mouse in juvenile mice treated at 1–2 months of age, with necropsy several days after treatment) and assessed with next-generation sequencing of the Rosa26-Q335X cassette. Across the dose groups, there was ≤1% indel mutagenesis at the target site. Panel C shows corrective adenine base editing of the CPS1 Q335X variant in lentivirus-transduced HuH-7 cells treated with k-abe. Editing was determined three days following treatment at the stated dose (concentration after dilution into cell medium). The best-fit agonist response curve with variable slope (4 parameters) and EC₅₀ and EC₉₀ values were calculated with GraphPad Prism. Panel D shows the evaluation of a high-priority subset of nominated off-target sites for any adenine-to-guanine editing via individual targeted amplicon sequencing in untreated cells versus cells 3 days following treatment with k-abe, at 1000 nanograms per milliliter of media, of four types: Q335X lentivirus-transduced HuH-7 cells and primary human hepatocytes (PHHs) from donor ICH (13 months of age, male), donor PDV (7 weeks of age, male), and donor YEQ (6 months of age, male). Out of 21 high-priority nominated off-target sites, data for only the 16 sites for which sequencing was successful are shown here.

Figure 3.. Biochemical Profile Before and After Treatment with K-abe.

Protein intake (panel A) and levels of plasma ammonia (panel B), glutamine (panel C), and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (panel D) during the patient’s lifetime, including the 7 weeks following k-abe dose 1 (up to day of life 256). The gray bars from left to right indicate periods of rotavirus-positive gastroenteritis prior to treatment, rhinovirus-positive upper respiratory infection after dose 1, and two viral illnesses after dose 2 (gastroenteritis followed by a new rhinovirus/enterovirus infection with associated viral transaminitis). The horizontal dotted lines indicate upper limits of normal laboratory value ranges. Panel E and panel F show pre-treatment and post-treatment plasma ammonia levels and urine orotic acid levels, respectively.