The focus of gene delivery research in the Davis Group is the synthesis and characterization of cyclodextrin-containing polycations (CDPs) that condense nucleic acids into complexes suitable for cellular uptake. We employ a systems approach to prepare complexes and modify them with stabilizing and targeting components that result in stable, well-defined DNA- or RNA-containing complexes (“polyplexes”) that are suitable for in vivo administration.
We have found that polycations containing cyclodextrin can achieve transfection efficiencies comparable to those of the best polycations reported while remaining essentially non-toxic. We have prepared a family of polymers that includes variations in charge spacing, charge type, and sugar type. A spacing of six methylene units between adjacent amidine groups within the comonomer gave the best transfection properties.
Gonazalez, H., Hwang, S.J., and Davis, M.E. New Class of Polymers for the Delivery of Macromolecular Therapeutics Bioconjugate Chemistry 6:1068-1074 (1999)
Hwang, S.J., Bellocq, N.C., and Davis, M.E. Effects of Structure of b-Cyclodextrin-Containing Polycations on Gene Delivery Bioconjugate Chemistry 12:280-290 (2001)
Cyclodextrins are differentiated by the number of sugar residues comprising the cyclodextrin ring. CDPs prepared with different cyclodextrin types showed little difference in gene delivery performance in vitro. However, the incorporation of beta-cyclodextrin reduced polycation toxicity much more than incorporation of simple sugars such as trehalose, suggesting that carbohydrate size affects toxicity. When the spacing between amidine charge centers and cyclodextrin moieties was increased, the transfection efficiency and toxicity of CDPs both increased. The toxicity-mediating effect of the cyclodextrin component appears tied to its interaction with the charge center. Further, quarternary ammonium polymers had consistently lower gene expression than their amidine analogs, suggesting that the charge center type plays a role in delivery efficiency.
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Reineke, T.M. and Davis, M.E. Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 1. Carbohydrate Size and Its Distance from Charge Centers Bioconjugate Chemistry 14:247-254 (2003).
Reineke, T.M. and Davis, M.E. Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 2. ChargeCenter Type Bioconjugate Chemistry 14:255-261 (2003).
Popielarski, S.R., Mishra, S., and Davis, M.E. Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 3. Cyclodextrin Type and Functionalization Bioconjugate Chemistry 14:672-678 (2003).
The unique structure of cyclodextrins allows us to modify CDP/DNA complexes by tethering a stabilizing polymer (poly(ethylene glycol) (PEG)) to a small molecule (adamantane, Ad) that forms strong, non-covalent inclusion compounds within the hydrophobic cyclodextrin cavity. This PEGylation is allosteric to the site of nucleic acid binding and endows the resulting polyplexes with stability in salt and serum that is essential for in vivo efficacy. Targeting ligands, such as galactose and transferrin, can be appended to Ad-PEG to allow preferential uptake of polyplexes in the desired target cells.
Pun, S.H. and Davis, M.E. Development of a Nonviral Gene Delivery Vehicles for Systemic Application Bioconjugate Chemistry 13:630-639 (2002).
Bellocq , N.C. , Pun, S.H., Jensen, G.S., and Davis , M.E. Transferrin-Containing Cyclodextrin Polymer-Based Particles for Tumor-Targeted Gene Delivery Bioconjugate Chemistry 14:1122-1132 (2003)
An improved understanding of gene delivery systems through rigorous physical characterization will aid efforts to improve these systems’ efficacy. We employ dynamic light scattering (DLS) and static, multi-angle light scattering (MALS) to quantify polyplex size and molar mass.
This information is combined with determination of bound vs. “free” polymer within polyplex formulations to calculate polyplex stoichiometry.
Isothermal titration calorimetry allows us to quantify the strength (K d) and amount (n) of Ad-PEG binding within our stabilized formulations.
The modifications necessary for in vivo efficacy of polyplexes can also affect their performance in unforeseen ways. Our efforts at characterization extend to exploring the intracellular mechanisms of nonviral gene delivery. As part of the systems approach in the Davis group, we have sought to identify and describe differences in the nature and performance of simple polyplexes and those modified to be more appropriate for physiological conditions. We have shown significant differences in the uptake and intracellular trafficking of unmodified and PEGylated polyplexes. Our laboratory emphasizes systems with in vivo applicability.
Mishra, S., Webster, P. and Davis, M.E. PEGylation Significantly Affects Uptake and Intracellular Trafficking of Non-Viral Gene Delivery Particles. Eur. J. Cell Biol. 83:97-111 (2004)
CDPs made in the Davis group are also highly effective for delivery of RNA-cleaving DNA enzymes (DNAzymes) and small, interfering RNA (siRNA) used to specifically down-regulate a target gene of interest.
Pun, S.H., Tack, F., Bellocq, N.C., Cheng, J., Grubbs, B.H., Jensen, G.S., Davis, M.E., Brewster, M., Janicot, M., Janssens, B., Floren, W., and Bakker, A. Targeted Delivery of RNA-Cleaving DNA Enzyme (DNAzyme) to Tumor Tissue by Transferrin-Modified, Cyclodextrin-Based Particles Cancer Biology & Therapy 3:7 (2004)
Polyplexes made with siRNA have been shown to down-regulate a target gene in vitro and in vivo. Co-transfection of pDNA and siRNA in cultured cells results in strong, sequence-specific down-regulation of the target gene. Luciferase-expressing Ewing sarcoma cells are injected into female NOD/scid mice and tumorogenesis is monitored non-invasively using a Xenogen camera (IVIS 100) system. SiRNA-containing polyplexes are targeted to the tumor via incorporation of transferrin (Tf), a protein whose cognate receptor (TfR) is highly upregulated on the surface of Ewing sarcoma cells. Low-pressure administration of these polyplexes via the tail vein can reduce tumor signal up to ~90%.
