Base and Prime Editing for Neurodevelopmental Epilepsy

Overview

Base editing and prime editing represent the latest generation of CRISPR-based gene editing technologies, enabling precise single-nucleotide changes in genomic DNA without inducing double-strand breaks (DSBs). For [neurodevelopmental epilepsies](/therapeutics/aav-gene-therapy-neurodevelopmental-epilepsy) (NDEs) caused by well-characterized point mutations — including [Dravet syndrome](/diseases/dravet-syndrome) ([SCN1A](/genes/scn1a)), [Angelman syndrome](/diseases/angelman-syndrome) ([UBE3A](/genes/ube3a)), and [KCNQ2 encephalopathy](/diseases/kcnq2-encephalopathy) — these technologies offer the potential for permanent, precise correction that addresses the root genetic cause[@rees2019].

Unlike traditional CRISPR-Cas9 (which cuts both DNA strands and relies on cellular repair pathways that are error-prone) or ASO approaches (which require repeat dosing and don't permanently alter the genome), base and prime editors make targeted, precise changes with minimal risk of off-target effects.

Base Editing

Mechanism of Action

Base editors consist of three components:

Overview

Base editing and prime editing represent the latest generation of CRISPR-based gene editing technologies, enabling precise single-nucleotide changes in genomic DNA without inducing double-strand breaks (DSBs). For [neurodevelopmental epilepsies](/therapeutics/aav-gene-therapy-neurodevelopmental-epilepsy) (NDEs) caused by well-characterized point mutations — including [Dravet syndrome](/diseases/dravet-syndrome) ([SCN1A](/genes/scn1a)), [Angelman syndrome](/diseases/angelman-syndrome) ([UBE3A](/genes/ube3a)), and [KCNQ2 encephalopathy](/diseases/kcnq2-encephalopathy) — these technologies offer the potential for permanent, precise correction that addresses the root genetic cause[@rees2019].

Unlike traditional CRISPR-Cas9 (which cuts both DNA strands and relies on cellular repair pathways that are error-prone) or ASO approaches (which require repeat dosing and don't permanently alter the genome), base and prime editors make targeted, precise changes with minimal risk of off-target effects.

Base Editing

Mechanism of Action

Base editors consist of three components:

Types of Base Editors

| Editor | Conversion | NDE Applications |
|--------|-----------|-----------------|
| CBE (Cytidine Base Editor) | C→T (G→A on opposite strand) | ~60% of disease-causing point mutations |
| ABE (Adenine Base Editor) | A→G (T→C on opposite strand) | ~20% of disease-causing point mutations |
| CGBE/AGBE | C→G (G→C) | Specific applications requiring GC changes |
| **Hypothetical dual base editing | Simultaneous C+T or A+G | Expandable targeting scope |

NDE-Specific Applications

SCN1A (Dravet Syndrome):

• ~40% of pathogenic SCN1A variants are missense mutations — many are C>T or A>G transitions addressable by CBEs or ABEs
• Examples: R1644H, W1204X, G1674E (all C>T candidates for CBE)
• Correction in patient-derived iPSCs has shown restoration of Nav1.1 channel function in neurons

• Some Angelman cases are caused by point mutations rather than deletions — these are prime candidates for base editing
• Restoring specific functional residues in UBE3A could reverse the Angelman phenotype
• Important: Must target only the paternal allele (avoiding the maternal deletion) — requires allele-specific design

• Gain-of-function mutations (causing self-limited neonatal epilepsy) vs. loss-of-function (causing KCNQ2 encephalopathy) — base editors could upregulate or correct as needed
• Correction of truncating mutations in KCNQ2 could restore functional KCNQ2 potassium channel subunits

Prime Editing

Mechanism of Action

Prime editing is the most versatile of the precision editing technologies, capable of making all 12 types of point mutations plus insertions and deletions, without DSBs or donor DNA templates[@anzalone2019].

The system has two components:

NDE-Specific Applications

SCN1A (Dravet Syndrome):

• Prime editing could correct all missense variants (not just transition mutations)
• Multi-kilobase insertions possible for larger SCN1A corrections
• Could be used to correct the precise variant in each patient, enabling personalized therapy

• The majority of pathogenic CDKL5 variants are point mutations addressable by prime editing
• Large deletions in CDKL5 could potentially be corrected with insertion editing

• Diverse mutation types (missense, nonsense, frameshift) — prime editing handles all
• Particularly valuable for nonsense mutations (premature stop codons) via insertion of wild-type sequence

PE3 vs PE5 System

| System | Mechanism | Efficiency | Off-target risk |
|--------|-----------|------------|-----------------|
| PE3 | Nick target strand, RT fills edit, cellular repair copies | Moderate | Lower |
| PE5 | Nick both strands, more efficient editing | Higher | Slightly higher |

Prime editing efficiency in neurons is typically lower than in dividing cells, requiring optimization of delivery, timing, and pegRNA design for post-mitotic neuronal applications.

Delivery Challenges

CNS Delivery of Editing Machinery

The key challenge for base/prime editing in NDEs is delivering the editing components to the right cells in the brain:

| Delivery Approach | Advantages | Disadvantages |
|------------------|-----------|---------------|
| AAV | Neuronal tropism (some serotypes), long-term expression | Cargo limit (~4.7kb) limits full editor + gRNA; immune response |
| LNP | Large cargo, scalable, redosable | Limited BBB crossing; transient expression |
| Exosome | Natural BBB crossing, low immunogenicity | Manufacturing challenges, variable yield |
| High-capacity adenovirus (HdAd) | Large cargo, long-term expression | Immune response, less neuronal tropism |

AAV delivery constraint: Most base editors (CBEs: ~5.3kb including SpCas9nick, ABEs: ~5.7kb) barely fit in AAV with a single gRNA. Strategies:

• Split-base editor systems (separate AAVs for each component)
• Use of smaller Cas9 orthologs (e.g., Cas12a, Cas9 from S. aureus)
• Minimized linker architectures

Cell-Type Specificity for NDEs

For SCN1A/Dravet, editing must be confined to GABAergic interneurons — editing in excitatory pyramidal neurons could worsen disease:

• Promoter-specific editors: Use GABAergic neuron-specific promoters (e.g., GAD1, GAD2) to drive editor expression
• Cell-type specific gRNA design: For allele-specific editing, exploit differences between mutant and wild-type alleles
• Transient delivery: Non-integrating delivery (mRNA + protein/RNP) to minimize off-targets

Off-Target Considerations

DNA Off-Targets

Both base and prime editors can cause unintended edits at genomic sites with partial homology to the target sequence:

• Whole-genome sequencing (WGS) is the gold standard for off-target detection
• DISCOVER-seq and CHANGE-seq provide unbiased off-target profiling
• Engineered deaminases (e.g., evoAPOBEC, TadA variants) show improved specificity

RNA Off-Targets (CBE-specific)

Cytidine base editors can cause widespread RNA editing (C-to-U conversions in mRNA), which was a significant concern with first-generation CBEs. New-generation CBEs (e.g.,evoAPOBEC, BE4max) have substantially reduced RNA off-targets through deaminase engineering[@grunewald2020].

Mitigation Strategies

Preclinical Data and Timeline

iPSC Models

Patient-derived iPSCs have been edited to model and test base/prime editing approaches:

• SCN1A iPSC models show that Nav1.1 dysfunction can be corrected by restoring wild-type sequence
• KCNQ2 iPSC models demonstrate that loss-of-function can be rescued by precise correction
• CDKL5 iPSC models show that editing can restore normal neuronal electrophysiology[@liang2023]

Animal Model Studies

• PE3 in mouse neurons: Prime editing in post-mitotic neurons has been demonstrated with ~10-20% efficiency using RNP electroporation[@chen2023]
• CBE in mouse brain: AAV-delivered CBE has edited neurons in vivo with low but detectable efficiency
• NHP studies: Prime editing in non-human primates is ongoing, with results expected 2026-2027

Timeline to Clinic

| Milestone | Expected |
|-----------|----------|
| Proof-of-concept in NHP models | 2026 |
| IND-enabling studies for NDE indication | 2027-2028 |
| First-in-human clinical trial | 2029-2030 |

Comparison of Editing Technologies

| Factor | Base Editing | Prime Editing | Traditional CRISPR-Cas9 |
|--------|------------|---------------|----------------------|
| Precision | Single nucleotide (C>T or A>G) | All 12 substitutions + indels | Requires DSB + homology |
| Efficiency | High | Moderate | High |
| Off-targets | Low (CBE: RNA concern) | Low-moderate | Higher (DSB-induced) |
| Cargo size | ~5kb | ~6kb+ | ~4kb |
| Indel formation | Minimal | Very low | Common |
| In neurons (in vivo) | Demonstrated | Demonstrated | Demonstrated |
| BBB crossing | Requires delivery optimization | Requires delivery optimization | Requires delivery optimization |
| NDE readiness | Earlier (simpler, higher efficiency) | Later (more versatile) | Mid-term |

Key Open Questions

Cross-Links

• [CRISPR Gene Editing](/technologies/crispr-gene-editing) — foundational technology page](/technologies)
• [Lipid Nanoparticle CNS Delivery](/technologies/lipid-nanoparticle-cns-delivery) — delivery platform](/technologies)
• [Exosome CNS Delivery](/technologies/exosome-cns-delivery) — delivery platform](/technologies)
• [AAV Vectors](/technologies/aav-vectors) — delivery platform](/technologies)
• [Gene Therapy for Neurodevelopmental Epilepsy](/technologies/gene-therapy-neurodevelopmental-epilepsy) — hub page](/technologies)
• [SCN1A Gene](/genes/scn1a) — Dravet syndrome target](/genes)
• [UBE3A Gene](/genes/ube3a) — Angelman syndrome target](/genes)
• [KCNQ2 Gene](/genes/kcnq2) — KCNQ2 encephalopathy target](/genes)
• [CDKL5 Gene](/genes/cdkl5) — CDKL5 deficiency target](/genes)
• [Stoke Therapeutics](/companies/stoke-therapeutics) — ASO competitor](/therapeutics)
• [Encoded Therapeutics](/companies/encoded-therapeutics) — CRISPRa competitor](/therapeutics)
• [GeneTx Biotherapeutics](/companies/genetx-biotherapeutics) — ASO competitor