Oral Presentation International Congress on Neuronal Ceroid Lipofuscinoses 2025

Engineered cell micropharmacies for in vivo production of TPP1 as enzyme replacement therapy in CLN2 Batten disease (130668)

Evan Kleinboehl 1 , Bryan J Jones 1 , Ethan M Niemeyer 1 , Luis Tecedor 1 , Beverly L Davidson 1 , Beau R Webber 2 3 4 , Branden S Moriarity 2 3 4 , Joseph G Skeate 2 3 4
  1. Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia Research Institute, , Philadelphia, PA, USA.
  2. Department of Pediatrics, University of Minnesota, , Minneapolis, MN, USA.
  3. Masonic Cancer Center, University of Minnesota,, Minneapolis, MN, USA.
  4. Center for Genome Engineering, University of Minnesota, , Minneapolis, MN, USA.

BACKGROUND: CLN2 disease, also known as late-infantile Batten disease, results from a deficiency of tripeptidyl peptidase 1 (TPP1), leading to progressive neurodegeneration. Current enzyme replacement therapy is carried out through repeated dosing using an Ommaya reservoir. Despite slowing disease progression this approach is burdensome and does not  reverse CNS pathology. As such, there is an urgent need for new treatment strategies. Here, we show our progress using a novel, non-virally engineered T cell micropharmacy platform for continuous, systemic in vivo enzyme delivery. We deployed a TPP1 expression transposon cassette under a constitutive MND promoter, coupled with a EGFR reporter, to track engineered primary T cells and provide a built-in enrichment and safety switch.

 

APPROACH: Human primary CD3⁺ T cells from multiple healthy donors were engineered with the TcBuster transposon system and demonstrated functional TPP1 secretion and co-expression of EGFRt. To enhance therapeutic efficacy of micropharmacy, we developed two additional approaches: a TPP1-APOE2 fusion to improve blood brain barrier penetration and an S1S3 co-expression strategy to increase mannose-6-phosphate tagging and lysosomal uptake.

 

RESULTS: T cell micropharmacies were successfully engineered from 4 donors and showed functional TPP1 secretion culture media. Cross-correction assays are currently underway to confirm uptake and functional rescue in TPP1-deficient cells as well as validation of second generation constructs. In addition, we are generating a novel NSG TPP1 knockout mouse model at the University of Minnesota, establishing a critical in vivo platform to enable upcoming preclinical studies.

 

FUTURE DIRECTIONS: Preclinical studies will be extended using the newly developed NSG TPP1 knockout model. We anticipate this animal model will provide a critical platform for human cell–based therapy testing and will be made available as a resource for the broader scientific community. Together, these advances position engineered T cell micropharmacies as a promising long-term enzyme replacement strategy for CLN2 disease.