TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

Mol Ther. 2024 Jun 5;32(6):1643-1657. doi: 10.1016/j.ymthe.2024.04.001. Epub 2024 Apr 6.

Abstract

Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

Keywords: gene therapy; hematopoietic stem cell; intron editing; lineage-specific expression; lysosomal storage diseases.

MeSH terms

  • Animals
  • CD11b Antigen / genetics
  • CD11b Antigen / metabolism
  • Cell Differentiation / genetics
  • Cell Lineage / genetics
  • Gene Editing* / methods
  • Gene Expression
  • Genetic Therapy / methods
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Hematopoietic Stem Cell Transplantation / methods
  • Hematopoietic Stem Cells* / metabolism
  • Humans
  • Iduronidase / genetics
  • Iduronidase / metabolism
  • Introns*
  • Mice
  • Mucopolysaccharidosis I / genetics
  • Mucopolysaccharidosis I / therapy
  • Myeloid Cells* / metabolism
  • Transcription Activator-Like Effector Nucleases* / genetics
  • Transcription Activator-Like Effector Nucleases* / metabolism
  • Transgenes*

Substances

  • Transcription Activator-Like Effector Nucleases
  • Iduronidase
  • Green Fluorescent Proteins
  • CD11b Antigen
  • enhanced green fluorescent protein