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Updated: Oct 14, 2025
Retroviral Transduction of T-cell Receptors in Mouse T-cells
Published on: October 22, 2010
Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8
Alexander Michels1, Annika M Frank2, Dorothee M Günther1,3
1Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, Germany.
Abstract:
Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes.
Insights
Engineered proteins targeting CD8+ cells improve gene delivery in mice using lentiviral (LV) and adeno-associated virus (AAV) vectors. This receptor targeting overcomes previous limitations for in vivo gene therapy applications.
Area of Science:
- Molecular Biology
- Immunology
- Gene Therapy
Background:
- Preclinical gene delivery in mice faces challenges with lentiviral (LV) and adeno-associated virus (AAV) vector efficiency.
- Lack of cell-type selectivity hinders in vivo gene therapy applications targeting specific lymphocytes.
Purpose of the Study:
- To develop novel targeting ligands for enhanced gene delivery to specific mouse immune cells.
- To improve the selectivity and efficiency of LV and AAV vectors for CD8+ T cell transduction.
Main Methods:
- Designed ankyrin repeat proteins (DARPins) were engineered to bind murine CD8.
- The CD8-binding DARPin was displayed on measles virus (MV) glycoproteins for LV and on AAV2 capsid (VP1) for AAV vectors.
- Gene delivery efficiency and cell-type specificity were evaluated in mouse splenocytes and blood.
Main Results:
- mCD8-LV demonstrated >99% selectivity for CD8+ lymphocytes and achieved 4-fold higher gene delivery than conventional LV.
- mCD8-LV mediated elimination of B lymphocytes and lymphoma cells in splenocyte cultures.
- mCD8-AAV (DART-AAV) showed >20-fold higher gene delivery efficiency and >99% specificity for CD8+ cells compared to standard AAV2.
Conclusions:
- Receptor-targeted DARPins significantly enhance gene delivery efficiency and CD8+ cell specificity for both LV and AAV vectors.
- This approach overcomes transduction barriers in mouse splenocytes, paving the way for improved in vivo gene therapy.
- DARPin-mediated targeting represents a promising strategy for precise immune cell manipulation in preclinical models.
