Related Experiment Videos
Adenoviral gene transfer is inhibited by soluble factors in malignant pleural effusions
R K Batra1, S M Dubinett, B W Henkle
1Department of Medicine and the UCLA/Wadsworth Pulmonary Immunology Laboratory, University of California at Los Angeles, Veterans Administration-Greater Los Angeles Health Care System, Los Angeles, California 90073, USA. rbatra@ucla.edu
Abstract:
Direct in vivo gene delivery is a prerequisite for many gene therapy strategies; however, efficacy has been limited by a lack of therapeutic gene transfer. In studying intrapleural malignancy as a model for the gene therapy of non-small cell lung cancer, we previously identified soluble chondroitin sulfate-proteoglycans/glycosaminoglycans (CS-PG/GAGs) in malignant pleural effusions (MPE) as factors that inhibit retroviral vector (RV) transduction. Similarly, we have observed inhibition to gene transfer in the fluid component of MPE using adenoviral (Ad) vectors. Analyses indicate that the factors responsible for the block are filterable, soluble, titrable, and heat stable (56 degrees C). Passage through microporous membranes fractionates the inhibitory factors into large (> 100 kD) components of the effusions. In contrast to RV transduction, hyaluronic acid or CS-PG/GAGs are not the inhibitors because the block is not reversed by pretreatment of the effusions with mammalian hyaluronidase, and exogenous addition of GAGs into the transduction media does not diminish Ad transduction. In considering the mechanism of action of the inhibitory factors, we observe that Ad entry, and specifically the binding of radiolabeled Ad to its target cell, is inhibited in the presence of MPE. Ad internalization may also be impaired; however, these studies exclude soluble fibronectin in MPE as a competitive inhibitor of Ad transduction. Lastly, sepharose A- mediated immunoglobulin depletion of MPE only partially reverses the block, and significant inhibition to Ad gene transfer persists at lower adenovirus:target cell ratios. Identifying the structural and functional basis for inhibition to Ad gene transfer may yield specific strategies to enable better in vivo translation of gene therapy approaches.
Insights
Malignant pleural effusions inhibit adenoviral gene transfer by blocking vector entry into target cells. Identifying these inhibitory factors is crucial for advancing gene therapy for lung cancer.
Area of Science:
- Oncology
- Gene Therapy
- Molecular Biology
Background:
- Direct in vivo gene delivery is essential for gene therapy, but its efficacy is hindered by limited gene transfer.
- Malignant pleural effusions (MPE) impede retroviral vector (RV) and adenoviral (Ad) vector gene transfer.
- Previous studies identified chondroitin sulfate-proteoglycans/glycosaminoglycans (CS-PG/GAGs) as inhibitors of RV transduction in MPE.
Purpose of the Study:
- To investigate the inhibitory factors in MPE that block adenoviral (Ad) vector-mediated gene transfer.
- To elucidate the mechanism by which MPE inhibits Ad gene transfer.
- To identify strategies for overcoming these inhibitory factors for improved in vivo gene therapy.
Main Methods:
- Analysis of MPE inhibitory factors, including filtration, heat stability, and fractionation (> 100 kD).
- Testing the role of hyaluronic acid and CS-PG/GAGs using hyaluronidase treatment and exogenous GAG addition.
- Assessing Ad vector entry and internalization inhibition, excluding soluble fibronectin.
- Evaluating the effect of immunoglobulin depletion on Ad gene transfer inhibition.
Main Results:
- MPE contains soluble, filterable, titratable, and heat-stable factors inhibiting Ad gene transfer.
- These inhibitors are distinct from hyaluronic acid and CS-PG/GAGs, as hyaluronidase treatment and GAG addition did not reverse the inhibition.
- MPE inhibits Ad vector binding to target cells, suggesting impaired entry.
- Immunoglobulin depletion partially reversed the inhibition, but significant inhibition persisted.
Conclusions:
- Malignant pleural effusions contain inhibitory factors that impair adenoviral vector gene transfer by blocking vector entry.
- These inhibitors are large molecular weight, soluble components distinct from GAGs.
- Understanding these inhibitory mechanisms is critical for developing effective gene therapy strategies for non-small cell lung cancer.