Gene therapy has the potential to permanently correct or prevent monogenic disorders such as cystic fibrosis. We have a demonstrated track record of utilizing many categories of viral and non-viral vectors to deliver genes to the airways;however, choosing the best gene therapy vector leads to a very significant dilemma. Non-integrating vectors may not persist and integrating vectors may cause insertional mutagenesis. There is a critical need for improved gene delivery tools that address this conundrum. Our long-term goal is to engineer a vehicle for gene delivery to cells that is a safe and effective therapeutic for cystic fibrosis. To this end, the goal for these proposed studies is to develop an integrating vector with a predictable integration pattern that targets 'safe harbor'genomic loci. Nonviral vector systems are used increasingly as tools for gene transfer applications. We successfully used the piggyBac DNA transposon system as an effective integrating vector for gene transfer. Method. Based on our preliminary studies, piggyBac transposase is amenable to modification. Furthermore, recent advances in the ability to engineer customized zinc finger proteins make the possibility of targeted transposition promising as a therapeutic approach. The overall hypothesis is that the piggyBac transposon system may be modified to retarget integration. We will use multiple approaches to direct the piggyBac transposase to designated loci, increase vector delivery efficiency, and improve the utility and safety of the vector for gene therapy. Here we propose to: 1) generate and validate the function of 'safe harbor'zinc finger protein/piggyBac transposase fusion proteins;2) map zinc finger protein/piggyBac transposase mediated transposon integrations in the genome;and 3) create a hybrid piggyBac/lentivirus vector system to improve delivery efficacy. These studies will provide important mechanistic information regarding the motifs important for directing piggyBac integrations.
The successful completion of these studies will bring us closer to the long-term goal of correcting the cystic fibrosis genetic deficiency. The specific goal of this project is to create a targetable transposon vector system for therapeutic gene transfer applications. Successful site-restricted transgene integration into human genomic DNA would have exciting and broad applications to the fields of gene therapy and molecular genetics.
|Sinn, P L; Hwang, B-Y; Li, N et al. (2017) Novel GP64 envelope variants for improved delivery to human airway epithelial cells. Gene Ther 24:674-679|
|Cooney, Ashley L; Abou Alaiwa, Mahmoud H; Shah, Viral S et al. (2016) Lentiviral-mediated phenotypic correction of cystic fibrosis pigs. JCI Insight 1:|
|Hornick, Andrew L; Li, Ni; Oakland, Mayumi et al. (2016) Human, Pig, and Mouse Interferon-Induced Transmembrane Proteins Partially Restrict Pseudotyped Lentiviral Vectors. Hum Gene Ther 27:354-62|
|Singh, Brajesh K; Hornick, Andrew L; Krishnamurthy, Sateesh et al. (2015) The Nectin-4/Afadin Protein Complex and Intercellular Membrane Pores Contribute to Rapid Spread of Measles Virus in Primary Human Airway Epithelia. J Virol 89:7089-96|
|Cooney, Ashley L; Singh, Brajesh K; Sinn, Patrick L (2015) Hybrid nonviral/viral vector systems for improved piggyBac DNA transposon in vivo delivery. Mol Ther 23:667-74|
|Mateo, Mathieu; Navaratnarajah, Chanakha K; Willenbring, Robin C et al. (2014) Different roles of the three loops forming the adhesive interface of nectin-4 in measles virus binding and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. J Virol 88:14161-71|
|Li, Xianghong; Burnight, Erin R; Cooney, Ashley L et al. (2013) piggyBac transposase tools for genome engineering. Proc Natl Acad Sci U S A 110:E2279-87|
|Oakland, Mayumi; Maury, Wendy; McCray Jr, Paul B et al. (2013) Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors. Mol Ther Nucleic Acids 2:e69|
|Li, Xiaopeng; Rossen, Nathan; Sinn, Patrick L et al. (2013) Integrin ?6?4 identifies human distal lung epithelial progenitor cells with potential as a cell-based therapy for cystic fibrosis lung disease. PLoS One 8:e83624|