Date: Thursday, Apr 18th
Presenter: Swati Mishra, Rutgers University
The design and development of non-viral gene therapies for the treatment of diseases and syndromes, where the molecular mechanism underlying the associated disorder is known, has made significant progress over the last decade. Efficient delivery of therapeutic gene is hindered by degradation in the systemic circulation as well as inadequate cellular entry and trafficking. In addition, failure to deliver gene drug into desired cells, thereby uptake of therapeutic gene by non-target cells lead to poor bio-distribution, low functional efficacy and toxicity.
In the present work, a multifunctional composite system for the efficient targeted intracellular delivery of antisense oligonucleotides (AONs) and short interfering RNAs (siRNA) is developed. Dioleoyl-3-trimethylammonium-propane (DOTAP) cationic liposomes were used to form complexes with nucleic acid sequences. Poly (propylacrylic acid) (PPAA), an anionic pH sensitive polymer, was incorporated into the lipoplex by electrostatic interactions in order to neutralize the overall charge of the nanoparticle and to mediate endosomal escape of the internalized oligonucleotides. We have designed and synthesized a family of copolymers in which poly (alkylene oxides) (PAO) are grafted onto PPAA to improve carrier stability in the bloodstream. Another panel of polymers was synthesized by conjugating hydroxyapatite binding ligand alendronate (ALN) to the grafted PAO chains to achieve active bone targeting. The composite Graplon (graft lipid-oligonucleotide nanocomplex) delivery system is thus comprised of a cationic lipid, nucleic acid sequence (AON or siRNA), and either PPAA, its graft copolymer with PAO (PPAA-g-PAO) or its ALN conjugated PAO graft analogue (PPAA-g-PAO-ALN). Polyethylene oxide (PEO) and polyetheramine (Jeffamine®) were selected as candidate PAO chains to achieve chemistries with varying degrees of lipophilicity to hydrophilicity.
Utilizing a panel of biophysical and delivery assays, we investigated chemistry-function relationships for this delivery system. A grafting extent of 1% was found to be sufficient for efficient gene silencing in cell cultures using anti-bcl2-AONs in ovarian cancer cells A2780 or anti-Runx2-siRNA in myo-progenitor cells C2C12, latter served as an in-vitro model to study the pathogenesis of heterotopic ossification. Delivery of siRNA targeting Runx2, a transcription factor downstream of BMP-2, to osteogenically stimulated C2C12 cells produced greater than 60% down-regulation of the Runx2 gene. This level of gene silencing was sufficient to inhibit alkaline phosphatase activity over the course of several days and calcium phosphate deposition over the course of 2 weeks. The DOTAP/siRNA/ PPAA-g-PAO-ALN nanocomplexes exhibited co-localization Cy-5 labeled siRNA at mineral- rich regions in BMP-2 stimulated C2C12 cultures.
These results demonstrate the feasibility of a biophysically guided approach to the delivery vector development and support the potential of the bio-conjugated Graplon formulation as a useful targeted delivery system for oligonucleotides in multiple applications.