Proven Intelligence Accelerating Next-Generation Therapies.

Executive Summary

For AAV-delivered gene editing programs, regulatory success hinges on a dual preclinical mandate: demonstrating potent on-target efficacy while rigorously profiling any off-target liabilities. A standard toxicology template is insufficient for these modalities. A tailored preclinical strategy is required to dissect the activity of the nuclease platform (e.g., CRISPR-Cas9, base editors) from the immunogenicity of the viral vector. This involves phase-appropriate assay development to quantify intended edits and sensitive screening for unintended genomic alterations, forming the core of a data package suitable for MHRA and FDA review.

The Dual Mandate: Quantifying Efficacy and Unintended Edits

The precision of gene editing platforms like CRISPR-Cas9, base editors, and prime editors presents a unique challenge for preclinical assessment. The objective is twofold: first, to confirm high-efficiency, durable editing at the intended genomic locus, and second, to conduct an exhaustive search for unintended edits across the genome.

The process requires a comprehensive characterization of the therapeutic candidate’s activity profile. This approach provides the necessary context for a thorough risk-benefit analysis by regulatory bodies.

A Tailored Preclinical Strategy for Gene Editing

A one-size-fits-all preclinical testing approach does not exist for these complex therapies. The strategy must be adapted to the specific nuclease platform, the AAV serotype used for delivery (e.g., AAV8, AAV9), the target indication, and the intended route of administration.

Our approach begins with designing studies that yield decision-making data. This includes establishing the dose-response relationship for on-target editing and defining the threshold for detecting off-target events.

“We started collaborating with UPenn Vector core in 2023 and the AAV vector which they manufactured laid a foundation for development of a gene therapy candidate which will enter soon preclinical studies. The key people from UPenn Vector Core joined Franklin Biolabs and our partnership transitioned without interruption from UPenn Vecor Core to Franklin Biolabs Research Vector Division. Franklin Biolabs is an essential collaborator in our AAV-vector based gene therapy candidate development and we hope to continue the partnership for years to come.”
— Biotech Partner

De-risking the Vector to Isolate Payload Effects

Accurate assessment of a gene editor’s specificity requires a clear biological window, free from confounding variables like a host immune response to the delivery vehicle. Before payload specificity can be properly evaluated, the effects of the AAV vector must be understood and managed.

Published data indicates that managing the innate immune response to the vector is a viable strategy. For instance, evidence suggests that transient immunosuppression can mitigate interferon responses to AAV administration (PMID: 35211641). By controlling for vector immunogenicity, we can more accurately attribute observed biological outcomes to the activity of the gene editing payload itself.

Methodologies for Comprehensive Assessment

A robust data package for an IMPD or IND submission requires orthogonal methods to build a complete picture of the editor’s activity.

• On-Target Efficacy Analysis:

  • Quantification of editing efficiency (e.g., indels, HDR) via next-generation sequencing (NGS).

  • Measurement of target protein restoration or knockdown using immunoassays or mass spectrometry.

  • Functional assays to confirm restoration of biological activity in the target tissue.

• Off-Target Liability Profiling:

  • In silico prediction to identify potential off-target sites with sequence homology.

  • Unbiased, genome-wide screens (e.g., GUIDE-seq, CIRCLE-seq) to empirically identify off-target cleavage sites.

  • Targeted deep sequencing of high-priority potential off-target sites in tissues from in vivo studies.

  • Full-service histology and digital image analysis to evaluate any tissue-level consequences.

This comprehensive approach provides the high-resolution data needed to advance novel gene editing therapies with a clear understanding of their performance and characteristics.