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Using Pulmozyme DNase treatment in lentiviral vector production
Aaron Shaw1, Daniela Bischof, Aparna Jasti
1Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Human Gene Therapy Methods
|March 21, 2012
Summary
Pulmozyme, a human DNase, effectively removes lentiviral vector DNA, matching or exceeding Benzonase performance without impacting vector infectivity. Early application reduces efficacy, suggesting post-production treatment is optimal for clinical-grade vector preparation.
Area of Science:
- Biotechnology
- Gene Therapy
- Molecular Biology
Background:
- Lentiviral vector production for clinical studies requires high purity.
- Benzonase, a bacterial DNase, is commonly used to remove contaminating DNA.
- An alternative DNase with potential clinical applications is sought.
Purpose of the Study:
- To compare the efficacy of Pulmozyme, a human DNase, against Benzonase for DNA removal from lentiviral vector preparations.
- To evaluate the impact of treatment timing (post-harvest vs. post-transfection) on DNA removal efficiency.
- To assess the effect of Pulmozyme on lentiviral vector infectious titer.
Main Methods:
- Lentiviral vectors were produced using HEK 293T cells and calcium phosphate transfection.
- DNA removal was assessed using quantitative PCR targeting the VSV-G plasmid.
- Pulmozyme and Benzonase treatments were applied at different time points and concentrations.
Main Results:
- Pulmozyme (20 U/ml, 1 hr) rendered plasmid DNA undetectable in the final vector product.
- Pulmozyme's effectiveness was equivalent or superior to Benzonase (50 U/ml).
- Early application of Pulmozyme post-transfection resulted in a 2-log DNA reduction, inferior to post-production treatment.
- Pulmozyme did not affect the infectious titer of the lentiviral vector.
Conclusions:
- Pulmozyme is an effective DNase for removing contaminating DNA from lentiviral vectors.
- Post-production treatment with Pulmozyme is more effective than early application.
- Pulmozyme is a viable alternative to Benzonase for clinical-grade lentiviral vector production, though cost implications should be considered.
