# Domain-substituted IGF2 tag modulates targeting of lentiviral gene therapy for Hunter syndrome

## TL;DR

A new IGF2-based tag called SWAP improves gene therapy for Hunter syndrome by targeting specific receptors while avoiding others.

## Contribution

The SWAP tag design allows modular receptor targeting while preserving lysosomal delivery and reducing off-target interactions.

## Key findings

- Deleting IGF2's central loop preserves CI-M6P/IGF2R binding but eliminates IR-A and IGF1R binding.
- Replacing the central loop with ApoE or RAP12 epitopes enables new receptor targeting like LRP-1.
- SWAP-tagged IDS gene therapy corrected Hunter syndrome pathology in multiple tissues in mice.

## Abstract

We present the SWAP design, a novel, structurally cohesive IGF2-based tag for modular receptor targeting during gene therapy for lysosomal storage disorders (LSDs). We found that IGF2’s central loop is critical for high-affinity binding to the insulin receptor (IR) and IGF1 receptor (IGF1R)—both involved in glucose homeostasis—but is not required for interaction with the cation independent mannose 6-phosphate/IGF2 receptor (CI-M6P/IGF2R)—a key target for lysosomal delivery. This formed the basis for designing the Substitution of the central-loop With Augmenting Peptides (SWAP) tag. By replacing the central loop with alternative epitopes, SWAP ensures high-affinity multimodal receptor targeting while maintaining structural integrity. In vivo, lentiviral gene therapy employing IDS fused to SWAP variants containing ApoE and RAP12x2 inserts corrected Hunter disease pathology across multiple tissues, including liver, spleen, heart, bone, and brain, matching the efficacy of the traditional IGF2 tag. These findings position SWAP as a novel and effective tag design for IGF2-based therapeutics with a more favourable ligand–receptor interaction.

The central loop of insulin-like growth factor 2 (IGF2) mediates binding to insulin receptor isoform A (IR-A) and IGF1 receptor (IGF1R) but is dispensable for interaction with CI-M6P/IGF2R. Here, we describe the design of the SWAP tag - an IGF2 analogue engineered to abolish IR-A binding, attenuate IGF1R binding, and retain high-affinity binding to CI-M6P/IGF2R - while also enabling modular targeting of additional receptors or proteins (e.g. LRP-1). This is relevant for all the IGF2-based therapeutics.

Deletion of IGF2’s central loop abolishes IR-A and IGF1R binding while preserving CI-M6P/IGF2R binding.Replacement of the central loop with alternative epitopes (e.g., ApoE or tandem RAP12) eliminates IR-A binding, reduces IGF1R binding, and confers new binding to receptors such as LRP-1.Fusion of SWAP tags to iduronate-2-sulfatase (IDS) - the enzyme deficient in Hunter syndrome - enhances correction of murine MPS II pathology compared to untagged IDS during lentiviral gene therapy.

Deletion of IGF2’s central loop abolishes IR-A and IGF1R binding while preserving CI-M6P/IGF2R binding.

Replacement of the central loop with alternative epitopes (e.g., ApoE or tandem RAP12) eliminates IR-A binding, reduces IGF1R binding, and confers new binding to receptors such as LRP-1.

Fusion of SWAP tags to iduronate-2-sulfatase (IDS) - the enzyme deficient in Hunter syndrome - enhances correction of murine MPS II pathology compared to untagged IDS during lentiviral gene therapy.

The central loop of insulin-like growth factor 2 (IGF2) mediates binding to insulin receptor isoform A (IR-A) and IGF1 receptor (IGF1R) but is dispensable for interaction with CI-M6P/IGF2R. Here, we describe the design of the SWAP tag - an IGF2 analogue engineered to abolish IR-A binding, attenuate IGF1R binding, and retain high-affinity binding to CI-M6P/IGF2R - while also enabling modular targeting of additional receptors or proteins (e.g. LRP-1). This is relevant for all the IGF2-based therapeutics.