N-of-1 CRISPR Therapy: Saving a Newborn

Introduction

News emerged in May 2025 from the Children’s Hospital of Philadelphia (CHOP) and Penn Medicine that a 6-month-old boy, referred to as KJ, received a personalized base-editing therapy correcting an ultra-rare mutation in his OTC gene. This gene encodes the liver enzyme ornithine transcarbamylase, critical for converting neurotoxic ammonia into urea. Absent treatment, hyperammonemia would lead to severe neurological injury or >50% mortality in infancy. Although long-term durability remains under observation, KJ’s rapid stabilization marks the first true N-of-1 CRISPR treatment.

Breakneck Breakthrough: Compressed Timeline

• Day 1–7: Whole-exome sequencing (Illumina NovaSeq 6000) identifies a c.386T>C point mutation in OTC. Bioinformatics pipeline (GATK, CADD scoring) confirms pathogenicity.
• Weeks 2–4: Primary patient fibroblasts cultured in DMEM/F12; CRISPR base editor (ABE8e fused to TadA deaminase) screened for gRNA efficiency using TIDE/ICE analysis.
• Month 2: In vitro optimization of lipid nanoparticle (LNP) formulation with ionizable lipid (DLin-MC3-DMA analog), DSPC, cholesterol, PEG-lipid, ~80 nm diameter (DLS measurement).
• Month 3: Generation of knock-in mouse model via pronuclear microinjection; on-target correction rate of 45% in hepatocytes (NGS at 5,000× coverage).
• Month 4: Pre-IND meeting with FDA’s Office of Tissues and Advanced Therapies (OTAT); IRB deliberation under expedited pathway (<21 CFR 312.20(b)).
• Month 5: Toxicology in C57BL/6 mice shows no significant off-target edits by CIRCLE-seq; whole-liver base editing at 42% yields normalized ammonia levels.
• Month 6: Non-human primate (cynomolgus) safety: single 1 mg/kg dose via IV infusion; no ALT/AST elevation, cytokine panel (IL-6, TNF-α) within baseline.
• Month 7: IND submission; FDA clearance in 7 days. KJ starts immunosuppression (tacrolimus) followed by three escalating LNP-delivered ABE8e doses.

Technical Architecture of the Base Editor and Delivery System

The base editor employed is ABE8e, an evolved adenine deaminase fused to a catalytically impaired Cas9 (SpCas9-HF1), codon-optimized for human hepatocytes. Guide RNAs were designed with AI-driven off-target prediction tools (DeepCRISPR) prioritizing minimal mismatches (≤2 mismatches outside seed region). mRNA transcripts are synthesized with CleanCap AG and 5′/3′ UTR elements for enhanced stability. LNPs employ a proprietary ionizable lipid sheath plus helper lipids in a molar ratio of 50:10:38:2 for DLin-MC3-DMA:DSPC:cholesterol:PEG-DMG respectively. Cryo-EM tomography confirmed uniform particle morphology at 80±5 nm.

Regulatory Innovation and Adaptive Oversight

In August 2025, the FDA published draft guidance “Considerations for N-of-1 Gene Therapy INDs”, codifying lessons from KJ’s case. The guidance endorses rolling data submissions, real-time pharmacovigilance, and streamlined IRB review under 21 CFR 56.104(d). “This adaptive framework bridges urgency with safety,” says Dr. Peter Marks, Director of CBER. The project leveraged the FDA’s OBA pilot program, reducing cycle times for protocol amendments to <5 days.

Preclinical Safety and Efficacy Assessment

• Mouse Model: HDR via CRISPR/Cas9 to introduce c.386T>C; on-target edit measured by ddPCR and Sanger sequencing.
• Off-Target Profiling: CIRCLE-seq and GUIDE-seq detected zero high-confidence off-targets; deep amplicon sequencing (2,000×) across top 20 sites.
• NHP Study: Single- and multiple-dose escalation in cynomolgus monkeys (up to 2 mg/kg). Biodistribution by qPCR and in situ hybridization confirmed 70% hepatocyte uptake; no hematological or biochemical toxicity.

Implications for Future Personalized Therapies

KJ’s therapy exemplifies a “platform technology” for individualized treatment, combining modular base editors and scalable LNP manufacture in GMP suites. Integration with cloud-based bioinformatics (AWS Batch, Nextflow) accelerates sequence design and QC reporting. Experts anticipate that this model will extend to G6PC, SMPD1, and other ultra-rare disorders. Ongoing research at Stanford and UTSW is exploring prime editing for similar N-of-1 applications.

Conclusion and Outlook

As KJ prepares for home discharge, follow-up includes quarterly ammonia challenge tests and liver MRI elastography. A 12-month cohort of N-of-1 therapies is already in IND review, reflecting a paradigm shift toward patient-specific genetic medicines. “This case validates decades of gene-editing research and paves the way for broader clinical adoption,” says Dr. Rebecca Ahrens-Nicklas.