Related Experiment Video
Updated: Feb 26, 2026
Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models
Published on: October 3, 2019
Targeting NSG Mice Engrafting Cells with a Clinically Applicable Lentiviral Vector Corrects Osteoclasts in Infantile
Ilana Moscatelli1, Henrik Löfvall1,2, Christian Schneider Thudium2
11 Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund University , Lund, Sweden.
Abstract:
Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by nonfunctional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to develop a clinically applicable lentiviral vector expressing TCIRG1 to correct osteoclast function in IMO. Two mammalian promoters were compared: elongation factor 1α short (EFS) promoter and chimeric myeloid promoter (ChimP). EFS promoter was chosen for continued experiments, as it performed better. IMO osteoclasts corrected in vitro by a TCIRG1-expressing lentiviral vector driven by EFS (EFS-T) restored Ca2+ release to 92% and the levels of the bone degradation product CTX-I to 95% in the media compared to control osteoclasts. IMO CD34+ cells from five patients transduced with EFS-T were transplanted into NSG mice. Bone marrow was harvested 9-19 weeks after transplantation, and human CD34+ cells were selected, expanded, and seeded on bone slices. Vector-corrected IMO osteoclasts had completely restored Ca2+ release. CTX-I levels in the media were 33% compared to normal osteoclasts. Thus, in summary, evidence is provided that transduction of IMO CD34+ cells with the clinically applicable EFS-T vector leads to full rescue of osteoclasts in vitro and partial rescue of osteoclasts generated from NSG mice engrafting hematopoietic cells. This supports the continued clinical development of gene therapy for IMO.
Insights
Gene therapy using a lentiviral vector (EFS-T) shows promise for infantile malignant osteopetrosis (IMO). This approach corrects osteoclast function by restoring the TCIRG1 gene, offering hope for treating this rare genetic disorder.
Area of Science:
- Genetics and Molecular Biology
- Hematology
- Orthopedics
Background:
- Infantile malignant osteopetrosis (IMO) is a rare, lethal autosomal recessive disorder.
- It is characterized by nonfunctional osteoclasts, often due to mutations in the TCIRG1 gene.
- The TCIRG1 gene encodes a subunit of the osteoclast proton pump essential for bone resorption.
Purpose of the Study:
- To develop a clinically applicable lentiviral vector for expressing TCIRG1 to correct osteoclast function in IMO.
- To compare the efficacy of two mammalian promoters: elongation factor 1α short (EFS) and chimeric myeloid promoter (ChimP).
Main Methods:
- Comparison of EFS and ChimP promoters for lentiviral vector expression.
- In vitro correction of IMO osteoclasts using a TCIRG1-expressing lentiviral vector (EFS-T).
- Transduction of patient-derived CD34+ cells with EFS-T and transplantation into NSG mice for in vivo assessment.
Main Results:
- The EFS promoter demonstrated superior performance compared to ChimP.
- In vitro, EFS-T corrected IMO osteoclasts restored Ca2+ release to 92% and CTX-I levels to 95% of control levels.
- In vivo, vector-corrected IMO osteoclasts derived from transplanted cells showed completely restored Ca2+ release and 33% of normal CTX-I levels.
Conclusions:
- Transduction of IMO CD34+ cells with the EFS-T vector leads to full in vitro rescue of osteoclast function.
- Partial rescue of osteoclast function was observed in vivo in mice engrafted with gene-corrected hematopoietic cells.
- These findings support the continued clinical development of gene therapy for infantile malignant osteopetrosis.
