Vehicles used for the delivery of therapeutic molecules such as DNA to cells must meet many criteria if they are to be administered systemically:

1. Non-toxic at effective dosages

2. Non-immunogenic

3. Promote efficient transfer of the therapeutic agent

Although viral vectors offer high efficiencies, researchers in the field are still grappling with a number of problems, e.g., the immunilogical response often caused by viruses. It is at the least inconvenient and possibly dangerous to administer drugs that cause an immune response.

Non-viral vectors typically are less efficient than their viral cousins, but they do not cause an immune response. Unfortunately non-viral gene delivery is often associated with significant toxicity, especially under conditions where they tend to function most efficiently.

In many cases, it is desirable to direct the transfer of the therapeutic agent not just through the cell membrane, but to a particular part of the cell. For instance, gene therapeutics should be directed to the nucleus. This task represents a further challenge for non-viral vector developers.

The Davis group has produced an intelligently designed cationic polymer that serves as an efficient non-viral vector, and is less toxic at effective dosages than its competition. It is a linear polymer containing difunctionalized beta-cyclodextrin monomers (AA) and other difunctionalized comonomers (BB) in the form AABBAABB. Cyclodextrin is found in FDA-approved drugs and is known to be non-toxic in many forms.

Reaction schemes for synthesis of beta-CD polymers. Polymer #4 has the most desirable qualities. You can see the reaction scheme for synthesis of the dicysteamine monomer in more detail by clicking here. Further details on these synthetic schemes can be found in the following references:

Gonzalez, H., Hwang, S.J., and Davis, M.E. (1999) New Class of Polymers for the Delivery of Macromolecular Therapeutics. Bioconjugate Chemistry 6, 1068-1074.

Gonzalez, H., Hwang, S.J., and Davis, M.E. (2000) Linear Cyclodextrin Copolymers. WO001734A1.

Davis, M.E., Gonzalez, H., and Hwang, S.J. (2000) Supramolecular Complexes Containing Therapeutic Agents. WO033885A1.

For information about how these polymers function as gene delivery vehicles, see the gene therapy link to the left.

Davis M.E.; Pun S.H.; Bellocq N.C.; Reineke T.M.; Popielarski S.R.; Mishra S.; and Heidel J.D. (2004) Self-Assembling Nucleic Acid Delivery Vehicles via Linear, Water-Soluble, Cyclodextrin-Containing Polymers. Current Medicinal Chemistry, 11, 179-197

Further work is in progress to improve the function of these polymers. For instance, it was found that the detailed structure of the polymer affects its performance for in vitro plasmid transfection.

CD polymers with y ranging from 4 to 6 and z=0,1 and x=1,2,3 were prepared and tested. The optimal polymer of these variants was z=1 and x=3. Notably, the maximum tolerable dose of this polymer in mice is between 100 and 200 mg/kg. Similar, non-CD polymers were formed as shown below, and were found to be extremely toxic.

Many features of the CD-containing polymers have been altered to develop a comprehensive understanding of the relationship between the structure and function of the polymer. Some of these features are the type of charge center, the type of carbohydrate and its functionalization, as well as the spacing of these elements relative to one another.

Hwang, S.J., Bellocq, N.C., and Davis, M.E. (2001) Effect of Structure of B-Cyclodextrin-containing Polymers on Gene Delivery. Bioconjugate Chemistry 12, 280-290.

Reineke, T.M. , and Davis, M.E. (2003) Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 1. Carbohydrate Size and Its Distance from Charge Centers. Bioconjugate Chemistry 14, 247-254.

Reineke, T.M. , and Davis, M.E. (2003) Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 2. Charge Center Type . Bioconjugate Chemistry 14, 255-261.

Popielarski, S.R., Mishra, S. , and Davis, M.E. (2003) Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 3. Cyclodextrin Type and Functionalization . Bioconjugate Chemistry 14, 672-678.

Of critical importance for any nonviral gene delivery system is that the polymer-DNA complex be stable in the conditions it will encounter in vivo. The Davis group has developed a method of stabilizing the cyclodextrin-polymer-DNA complexes by contacting the complexes with short PEG molecules that have been functionalized on one end with adamantane. The other end of the PEG molecule may be left inert, or it can be functionalized with a targeting agent such as galactose, below.

The adamantane forms an inclusion complex with the cyclodextrin cups of the polymer, leading to the formation of complexes where the positive charge is masked, inhibiting aggregation.

Below, in A, the top lines show the conventional CDP-DNA complexes aggregating in salt solution, with and without non-adamantane-containing PEG present. The bottom line is the fully formulated particles, which aggregate much more slowly. In intermediate effect is observed with an intermediate amount of adamantane-PEG.

This concept has been extended to targeting with the protein Transferrin for tumor therapy. The Transferrin-PEG complexes carrying DNA coding for luciferase demonstrated increased gene expression over untargeted complexes, and this increased uptake could be attenuated by the addition of free Transferrin.

Pun, S.H., and Davis, M.E. (2002) Development of a Nonviral Gene Delivery Vehicle for Systemic Application. Bioconjugate Chemistry 13, 630-639.

Bellocq, N.C., Pun, S.H., and Davis, M.E. (2003) Transferrin-Containing, Cyclodextrin Polymer-Based Particles for Tumor-Targeted Gene Delivery. Bioconjugate Chemistry 14, 1122-1132.