Abstract
Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood-brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques.
Overview
- The study aims to develop a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA) for efficient tumor-selective delivery of nucleic acid therapeutics to diseased sites via clinical systemic routes. The study focuses on the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. The primary objective of the study is to provide a detailed protocol for the production and control of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. The study utilizes a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing.
Comparative Analysis & Findings
- The study compares the outcomes observed under different experimental conditions or interventions detailed in the study. The study identifies significant differences in the results between the TPA particles and existing M13 phage vectors. The TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The study discusses the key findings of the study and how they relate to the initial hypothesis. The study demonstrates that the TPA particles are more efficient in delivering nucleic acid therapeutics to diseased sites via clinical systemic routes compared to existing M13 phage vectors.
Implications and Future Directions
- The study's findings have significant implications for the field of research or clinical practice. The study identifies limitations of the study that need to be addressed in future research. The study suggests possible future research directions that could build on the results of the study, explore unresolved questions, or utilize novel approaches. The study highlights the importance of developing efficient tumor-selective vectors via clinical systemic routes for the delivery of nucleic acid therapeutics to diseased sites. The study suggests that future research should focus on improving the efficiency and specificity of TPA particles for tumor targeting and delivery of nucleic acid therapeutics. The study also suggests that future research should explore the use of TPA particles for the delivery of other types of nucleic acid therapeutics, such as RNAi and CRISPR-Cas9.