DNA delivery to immune cells via a novel antibody targeted system

Shah, Neha D. (2011). DNA delivery to immune cells via a novel antibody targeted system PhD Thesis, School of Pharmacy, The University of Queensland.

       
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Author Shah, Neha D.
Thesis Title DNA delivery to immune cells via a novel antibody targeted system
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
Publication date 2011-03
Thesis type PhD Thesis
Supervisor Harendra S. Parekh
Raymond J. Steptoe
Total pages 336
Total colour pages 19
Total black and white pages 317
Subjects 111504 Pharmaceutical Sciences
110703 Autoimmunity
270801 Gene Therapy
Formatted abstract
T-cell mediated autoimmune diseases, such as Type 1 Diabetes, occur due to breakdown in T-cell tolerance resulting in autoimmune attack of healthy tissues. For T-cell mediated autoimmune diseases, restoration of tolerance in the T-cell compartment is an important therapeutic goal. Antigen presenting cells (APC) play an important role in maintaining self-tolerance by inactivating self-reactive T-cells.

Therapeutically, tolerance can be restored in the T-cell compartment by expressing antigens (Ags) genetically-targeted to APC (B cells and dendritic cells). There are a variety of ways to enable such targeted expression. For example, transgenic mice can be constructed in which a transgene is expressed under an APC-specific promoter. An alternative approach might be to use a viral vector to transduce hematopoietic stem cells that can then be transplanted to a recipient. Viral vectors are highly effective for gene transfer; however they have several drawbacks for use in clinical systems. Therefore, for application of genetically-targeted Ag expression as a tool for tolerance induction in a clinical setting, the development of safe and effective non-viral vectors may represent a significant breakthrough.

Dendrimer technology is an emerging tool for the safe and effective delivery of genes to cells. Conventional, spherical dendrimers comprise symmetrically branched polymers ending with active terminal functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, high generation (G4-G8) PAMAM dendrimers exhibit significant toxicity due to their high cationic charge density and this has limited their application in vivo. Another limitation arises when attempting to site-specifically attach targeting-ligands to these spherical systems, as all surface active groups have comparable reactivity, making this feat very difficult. Therefore, low-generation asymmetric dendrimers, which are likely to be less toxic, and to which site-specific incorporation of targeting-ligands could be achieved presents a viable alternative to currently-available spherical dendrimers.

With this in mind, a series of peptide-based asymmetric dendrimers were synthesised using solid phase peptide synthesis (SPPS) and an orthogonal protecting group strategy. The asymmetric dendrimers constructed were compared to spherical PAMAM G1 dendrimers of comparable charge for their ability to complex plasmid DNA (referred to as DNA in this thesis). A monoclonal antibody (mAb) was selected as targeting ligand. These conjugates were then complexed with DNA and targeted to cells in vitro to assess DNA delivery.

The results presented in this thesis, show successful asymmetric dendrimer synthesis as confirmed by RP-HPLC, ESI±-MS and 1D & 2D NMR, with dendrimers obtained at high purity and yield. It is also shown that the asymmetric dendrimers can complex DNA into stable toroids, which have been reported as necessary for efficient cell transfection. It was also established that the asymmetric dendrimer/DNA complexes were minimally cytotoxic. More importantly, the mAb-dendrimer conjugates targeted B-cells and delivered DNA in vitro to primary cells. This paves the way for these systems to be tested in vitro to achieve ~40 – 50 % transfection efficiency and then tested in vivo for their ability to restore tolerance.
Keyword Low-generation peptides
Asymmetric dendrimers
Toroids
Cytotoxicity
Tolerance
B-cells
Antibody reduction
Gene delivery

 
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Created: Sun, 24 Jul 2011, 00:38:04 EST by Ms Neha Shah on behalf of Scholarly Communication and Digitisation Service