Development and understanding of nucleic acid nanoparticles for human gene therapy

Gene therapy and/or repair remains a viable and attractive option for a large number of childhood diseases, both inherited and acquired. We not only tackle the correction of the primary defect in CF, dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR), with our gene delivery program, but we also address the correction of secondary defects such as susceptibility to infection and exaggerated inflammatory responses. Many of these secondary defects are present in other disease of the lung, liver, and brain, and thus, our studies have also extended beyond just targeting the CF lung.

In our laboratory, we use nanotechnology to deliver genes and/or other nucleic acids to target cells in the lung, liver, eye, or brain to effect therapeutic gene expression or repair mutated genes. These organs are targets for a variety of diseases including cystic fibrosis (lung and liver), hemophilia (liver), macular degeneration (eye), and Parkinson's Disease (brain). Our nucleic acid nanoparticles (NNP) are complexes of DNA or RNA with carrier proteins that are 100-200 nanometers in size (Figure 1). These particles can enter the cell and deliver genes for expression in the nucleus or RNA to the cytoplasm, and can be used to affect gene therapy or repair on target cells, depending on disease indication (Figure 2).

The goal of our current studies is to examine the safety, efficacy, immunogenicity, and the cellular biology of various formulations of NNPs. Although NNPs have been tested and found to be safe and efficacious in humans, gene delivery is low.

Figure 1. Schematic of PEG-PolyK DNA nanoparticle design.

Figure 2. Assessment of impact of promotor on DNA nanoparticle mediated gene expression.  Wild type C57BL/6 mice received 100 µg of compacted pCMVluc or pUbBluc by tracheal administration and gene expression was monitored by BLI (n = 5 for each group). Imaging of mice dosed with pCMVluc was conducted at days 2 (A), 6 (B), and 14 (C).  For mice that received pUbBluc, imaging was conducted on days 2 (D), 6 (E), 14 (F), 35 (G), 42 (H), 49 (I), and 56 (J). Panel (K) demonstrates total emitted photons/s for each mouse throughout the experimental time course.

Next Steps

Improving the in vivo efficiency of NNPs is logical and necessary for advancement to gene therapy application in humans. Two of the major determinants of efficient NNPs delivery in vivo are:

1. The escape of intact particles from the extracellular milieu encountered upon administration (airway surface liquid and mucus for transtracheal or blood for intravenous administration)
2. The interaction of the particles with cellular proteins that facilitate uptake and transfection.

A number of researchers are actively working on understanding the determinants of escape from the extracellular compartment, and this pursuit is presently satisfied in the field. For example, we and others have conjugated NNPs to polyethylene glycol to enhance stability and decrease toxicity in vivo. However, examination of the determinants of cellular uptake has been limited to identification of the role of nucleolin as a cell-surface receptor for the particles. Therefore, it seems logical to us to conduct studies in systems where mechanisms of uptake can be examined. To do this, we plan to study primary cells (a more clinically relevant cell-culture system) and validate our results both in primary cells from mice and in vivo.

Our DNA nanoparticles (co-invented by Dr. Ziady) have set a paradigm for non-viral gene delivery in vivo. Our lab has collaborated with Dr. Zhenghong Lee at Case Western Reserve University (CWRU) and Dr. David Yurek at the University of Kentucky to develop imaging techniques that allow us to examine the activity and distribution of this gene delivery agent in vivo using real time measurements. We have made inroads in translating these experiments to human studies. These studies have allowed us to more efficiently examine the effects of repeated administration over time, as this is needed for a non-viral and non-integrating gene expression system.

Furthermore, in recent years we have focused on the biology of the DNA nanoparticles in cells by examining the interactome of the particles. We have defined a number of proteins and cellular pathways responsible for the uptake and nuclear delivery of the particles. This is the first time the biology of the particles has been examined. Our plan is to better understand the determinants of successful gene delivery by the particles and manipulate these pathways to enhance the utility of this vector in humans. The long-term goal for the gene therapy work in our lab is to develop DNA nanoparticles that can target specific cell types, including respiratory airway epithelia, hepatocytes, or neurons.