Understanding and overcoming the intracellular barriers associated with nonviral nucleic acid delivery to neurons

Nucleic acid-based therapies offer the potential to revolutionize the treatment of neurological disease and spinal cord injury. Clinical implementation of nucleic acid-based therapies will require vectors that safely and efficiently mediate delivery to targeted neurons. While select viral vectors demonstrate efficient delivery to neurons, their utility as therapeutic agents is complicated by safety concerns and limitations associated with large-scale viral production. Nonviral vectors could potentially overcome these limitations, but current formulations yield unacceptably low delivery efficiencies to neurons, in part due to their poor intracellular delivery characteristics. Improved nonviral vectors must be designed to efficiently navigate through the intracellular milieu by overcoming a series of intracellular barriers.; This dissertation is based on the premise that efficient nonviral nucleic acid delivery to neurons can be achieved through a rational design process. Part I establishes a detailed understanding of the intracellular barriers encountered by nonviral vectors delivered to neurons. In Chapter 2, methods are described for distinguishing between surface-bound and internalized materials associated with neurites using an environmentally sensitive fluorophore. Chapter 3 describes the application of a microfluidic, compartmented neuron culture system to localize the delivery of vectors to cell bodies or neurites for analysis of the intracellular transport properties of internalized vectors. These techniques are implemented in Chapter 4 to quantitatively compare the major intracellular barriers encountered by lipid- and polymer-based vectors in neurons.; Guided by insights into the major intracellular barriers encountered by nonviral vectors in neurons, Part II explores peptide-based strategies to enhance the efficiency of intracellular delivery by nonviral vectors. Particular attention is paid to peptides that mediate attachment to intracellular motors for assisted intracellular trafficking as well as peptides that facilitate endosomal escape. In Chapter 6, a peptide is evaluated for its ability to attach artificial cargo to the dynein motor complex for motor-assisted transport toward the nucleus. In Chapter 7, a peptide containing domains for both dynein binding and endosomal escape is evaluated for its ability to confer each of these properties on nonviral vectors. Finally, recommendations for future areas of study are offered in Chapter 8.