Polymeric Nanoparticles for Nucleic Acid Delivery

Transcription

1 Polymeric Nanoparticles for Nucleic Acid Delivery Jordan J. Green, Ph.D. Biomedical Engineering, Ophthalmology, & Neurosurgery Johns Hopkins University July 9, 2014

2 Disclosures Potential conflicts of interest are managed by the Johns Hopkins Medical Institutions Committee on Outside Interests

3 Polymeric Nanoparticles for Intracellular Delivery Deliver sensitive biological cargos like proteins, peptides, DNA, & RNA Can potentially cure a disease, not just treat symptoms: Genetic Diseases Acquired Diseases

4 Viral Gene Therapy Tragedies Nature Med. 6(1) 2000, Nature Med. 8(11) 2002, Nature , Nature Med. 13(9) 2007

5 Advantages of Polymers for Gene Therapy Safety Minimal immune response Minimal cellular toxicity Not carcinogenic Large cargo capacity Flexibility to target different cells, tissues Low resistance to repeated administration Ease of production and quality control

6 Non-viral Gene Delivery is Ineffective Gene Expression 100 % Positive Nrml. Expression H 2 N N H N NH N H N HN H N H 2 N NH N NH N NH 2 0 Polyethylenimine 25k PEI Ad, MOI=100 Ad, MOI = 500 Polyethylenimine (PEI) is the gold standard Adapted from Green et al. Adv Mat 2007

7 Mechanism of Intracellular Delivery Sunshine, Bishop, & Green, Therapeutic Delivery 2011

8 Hydrolytic Poly(Ester Amine)s and Reducible Poly(Amido Amine)s One step reaction: amine monomer conjugate addition to diacrylates or diacrylamides High-throughput synthesis: Unique polymers, high diversity No purification needed: Synthesis neat or in DMSO No byproducts, no side reactions Tunable polymer function Green, Annals BME 2012

9 Biomaterial Libraries 1,000+ polymers in library and growing Structural diversity Tune cargo binding and release Tune degradability Tune cell-material interactions Non-cytotoxic Bhise et al. Biomaterials 2010

10 Gene Delivery Nanoparticles DNA/RNA Delivery Virus-like efficacy Non-viral advantages Human Umbilical-Vein Endothelial Organoid Retinal Neuron mes Retinal Endothelial Human Embryonic Stem Cells Organoid Glia

11 Plasmids per Particle Bhise et al. Small 2012

12 Cancer vs. Non-cancer: Liver Tzeng et al. Journal of Biomedical Materials Research (Part A) 2013

13 Cancer vs Non-cancer: Liver

14 Polymer 457 (50 w/w) Tzeng et al. Journal of Biomedical Materials Research (Part A) 2013

15 Polymer 457 (75 w/w) Tzeng et al. Journal of Biomedical Materials Research (Part A) 2013

16 Polymer 537 (w/w) Tzeng et al. Journal of Biomedical Materials Research (Part A) 2013

17 Lyophilized Nanoparticles are Stable Efficacy retained at -20C for at least 2 years

18 Brain Cancer Strategy Guerrero-Cazares et al ACS Nano 2014

19 Specificity to Brain Tumor Initiating Cells Guerrero-Cazares et al ACS Nano 2014

20 Application to Brain Cancer Patient derived glioblastoma (GB) High transfection of 3D GB neurospheres matches 2D monolayer Athymic mice intracranially injected with human GB 9 weeks later injected with NPs Lyophilized particles maintain efficacy for >2 years Guerrero-Cazares et al ACS Nano 2014

21 Application to Lung Cancer Small Cell Lung Cancer Model (subcutaneous H446 xenograft) TP53 often mutated Exogenous expression of TP53 caused apoptosis and reduced tumor growth Kamat et al. Molecular Cancer Therapeutics 2013

22 Application to Ophthalmology Subretinal Injection 1 µg / 1 µl Choroid flatmount 72 hrs injection mrna Protein Fluorescence Non-cytotoxic Sunshine et al. PLoS ONE 2012

23 sirna Delivery Nanoparticles Scrambled sirna 4310, 1.2:1 12 days after transfection GFP sirna Knockdown after 12 days (4310) Tzeng et al. Advanced Healthcare Materials 2013

24 Bioreducibility Reduces Cytotoxicity and Improves Knockdown Kozielski et al. Chem Comm 2013

25 Intermediate Bioreducibility is Optimal Kozielski et al. ACS Nano 2014

26 Cancer-specific Killing via sirna Delivery

27 Conclusions A polymer library approach allows for enabling technologies such as intracellular delivery of DNA and sirna Applications for Cancer, Regenerative Medicine, Ophthalmology, and Immunology

28 Acknowledgements Dr. Nupura Bhise Corey Bishop Jayoung Kim Kristen Kozielski Ron Shmueli Joel Sunshine Dr. Stephany Tzeng Dr. Peter Campochiaro Dr. Warren Grayson Dr. Christine Hann Dr. Aleksander Popel Dr. Jonathan Schneck Dr. Don Zack Dr. Hugo Guerrero-Cazares Funding: Dr. Alfredo Quinones-Hinojosa NIH (NIBIB, NCI), TEDCO MSCRF, INBT