RNA-NANOSPHERES FOR CONTROLLED GENE DELIVERY SYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0191129
• Project Start Date: Oct 1, 2001
• Project End Date: Oct 1, 2006
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
BIOLOGICAL & ENVIRONMENTAL ENGINEERING

Non Technical Summary
Gene delivery is an important and indispensable tool for both research and medicine. Current DNA-based gene delivery systems are inefficient and often toxic. The overall objective of this proposal is to develop an efficient, long-term, and low-cost gene delivery system that is based on RNA-encapsulated biocompatible and biodegradable polymeric nanospheres.

Animal Health Component

50%

Research Effort Categories

Basic

30%

Applied

50%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	7010	1040	30%
305	7299	1040	30%
404	7010	2020	20%
404	7299	2020	20%

Knowledge Area
305 - Animal Physiological Processes; 404 - Instrumentation and Control Systems;

Subject Of Investigation
7010 - Biological Cell Systems; 7299 - Research equipment and methods, general/other;

Field Of Science
1040 - Molecular biology; 2020 - Engineering;

Keywords

gene therapy

controlled release

microspheres

dna

rna

transfection

delivery systems

biodegradation

production systems

drugs

genetic engineering

animal physiology

cell biology

molecular biology

agricultural engineering

gene structure

gene function

production efficiency

process development

optimization

cytosol

endoplasmic reticulum

new technology

microscopy

product evaluation

Goals / Objectives
The ability to deliver genes into animal or plant cells is extremely important. For example, gene delivery has played a major role in protein-based medicine (i.e., productions of large-scale therapeutic proteins). Gene delivery has also become a powerful and popular research tool for elucidating gene structure and functions. In addition, gene delivery will be indispensable in developing new approaches for future gene-based medicine. Current gene delivery systems, however, are very inefficient, mainly due to the three major barriers along the gene delivery pathway: low uptake across plasma membrane, less stability inside the cytosol, and nuclear targeting. Therefore, developing efficient gene delivery systems is urgently needed. Almost all of the gene delivery systems developed so far are DNA-based delivery systems. This is expected since DNA molecules are carriers of genes. But the goals of most gene delivery systems are usually not genes themselves, but gene products "C proteins coded by delivered genes. Therefore it is conceivable that one can achieve gene delivery by delivering not DNA, but RNA, which will effectively bypass the cell nucleus since proteins are synthesized inside cytosol from mRNA. The major problem of this hypothesis has been the instability of RNA (both outside and inside of a cell), which also makes large preparation of RNA very difficult. With the advancement of controlled release polymers, it is proposed that polymeric nanospheres can shield RNA molecules from environmental factors before delivery and protect RNA molecules from degradation before release. There are three specific aims in this proposal. First, to explore methods and optimize conditions of encapsulating RNA molecules into biocompatible/biodegradable polymeric nanospheres. These RNA-nanospheres will totally shield RNA from the environment before delivery thus effectively increasing RNA stability. Second, to deliver RNA-nanospheres to the cytosol. Such delivery will release RNA intracellularly in a controlled and sustained fashion, ultimately prolonging the protein expressions. Third, to conjugate endoplasmic reticulum (ER) targeting signals to the nanospheres for precise transport regulation. ER targeting should be easier than nuclear targeting because of the larger surface area and fewer obstacles of ER membranes. The overall objective of this proposal is to develop an efficient, long-term, low-cost gene delivery system that is based on RNA and biocompatible/biodegradable polymers. There are many advantages of using RNA over DNA for gene delivery. First the destination of the delivery is the cytosol, not the nucleus. Cytosol delivery is by far easier and more efficient than nucleus delivery. Second, transcription process is not needed for RNA delivery, which reduces complicated cellular responses. Third, host-genome integration is not possible for RNA delivery, which minimizes long-term side effects. There are also numerous advantages of employing polymer-mediated RNA delivery over conventional delivery methods, which include predictable, site-specific, long-term, and sustained delivery.

Project Methods
The general approach of the proposed research will employ molecular bioengineering principles and methods. Specifically, the proposal is divided into three major stages according to specific aims: fabrication, delivery and modification. In stage 1, the main focus is to fabricate RNA-microspehres in vitro. Two types of techniques are proposed: chemical fabrication and nanofabrication. In the chemical approach, double emulsion methods will be employed and optimized with several biocompatible/biodegradable polymers including FDA-approved PLA and PLGA. In the nanofabrication approach, micron-sized hydrophobic and hydrophilic islands will be patterned on a silicon wafer using photo-lithography (for masks) and soft-lithography (for stamps). Various patterning reagents will be tested and evaluated. Scanning electron microscopy and light scattering measurement will be employed to assess the morphology and the size of fabricated RNA-nanospheres. In vitro release curves with different RNA loadings and different polymers will be constructed by incubating RNA-nanospheres in RNAse-free buffer. The integrity as well as the degradation of RNA molecules will be evaluated by electrophoresis. Bulk RNA (total RNAs from tissues, not just mRNA) will be tested initially in this stage, which will significantly reduce the cost of raw materials. In stage 2, the main focus is to deliver RNA-nanospheres intracellularly. Once the conditions of RNA-nanospheres fabrication are optimized (stage 1), the RNA will be labeled by fluoro-probes. Microinjections will be applied first to quickly test the feasibility of intracellular controlled release of RNA via RNA-nanospheres. Endocytosis pathways will be explored later, such as using endosomatrophic reagents, to facilitate the uptake of RNA-nanospheres. Microfabricated devices, such as micro-guides, which will facilitate high-throughput microinjections, may also be used as alternatives if all other methods fail. Chinese Hamster cells (CHO) will be used as a model system. The delivery efficiency will be assessed by quantitative fluorescence microscopy. Cytotoxicity will also be determined. In stage 3, the main focus is to modify RNA-nanospheres. Two types of modifications are proposed. First, to conjugate a peptide transduction domain (PTD), such as HIV-tat proteins, to PLGA/PLA molecules so that RNA-nanospheres can be pulled through plasma membrane without endocytosis. Second, to crosslink an ER signal to RNA molecules so that encapsulated RNA can be targeted to ribosomes. Modified RNA (via nucleic acid crosslinking chemistry) and/or modified PLGA/PLA polymers (via chemical conjugations) will be incorporated into RNA-nanospheres. These modifications are expected to enhance total delivery efficiency.

Progress 10/01/01 to 10/01/06

Outputs
Gene delivery is an important and indispensable tool for both research and medicine. Current DNA-based gene delivery systems are inefficient and often toxic. The overall objective of this proposal is to develop an efficient, long-term, and low-cost gene delivery system that is based on RNA-encapsulated biocompatible and biodegradable polymeric nanospheres. Towards that end, we have teamed up with Prof. Mark Grinstaff from Boston University and used a charge-reversal amphiphiles for both DNA and RNA delivery. The charge reversal amphiphiles are a new type of lipids that shows great promise in drug delivery. These synthetic vectors transforms from a cationic to an anionic amphiphile intracellularly. Enhanced gene transfection was observed using these vectors compared to current cationic amphiphiles. The delivery of siRNA was also performed in several cell lines and enhanced gene knockdown was observed.

Impacts
The charge-reversal amphiphile system may provide a valuable alternative to both DNA and RNA delivery.

Publications

• Prata, C.A.H., P. Barthelemy, Y. Li, D. Luo, T.J. McIntosh, Lee S.J. and M.W. Grinstaff. 2006. Charge-resersible lipids for DNA delivery. FASEB Journal 20 (4):A73-A73.
• Luo, D. and W.M. Saltzman (invited authors). 2006. Thinking of Silica. Gene Therapy 13(7):585-586.
• Prata, C.A.H., Y. Zhao, P. Barthelemy, Y. Li, D. Luo, T.J. McIntosh, S.J. Lee and M.W. Grinstaff. 2004. Charge-reversal amphiphiles for gene delivery. J Am Chem Soc 126(39):12196-7.

Progress 01/01/05 to 12/31/05

Outputs
We have started a new system for gene delivery; branched DNA is used as the delivery vector instead of encapsulation naked DNA/RNA. Using DNA as a scaffold material makes it possible to deliver multiple genes as well as multiple entities such as DNA plus RNA. The conjugation steps have been carried out and optimized. We have also started to use Nickel and 6xHis tags to attach proteins to the DNA framework in order to improve the efficiency of controlled gene delivery systems.

Impacts
Using DNA as a delivery vector can potentially increase not only the delivery efficiency but also the pay-load capacity of gene delivery systems. More importantly, it could make multi-gene/multi-entity delivery possible (e.g., RNA plus DNA).

Publications

• Li, Y. and Luo, D. 2005. (invited author), High Throughput Codes For Molecular Detections: From Millimeter to Nanometer. BioForum Europe, Dec. 2005.
• Stavis, S.M., J.B. Edel, Y. Li, K.D. Samiee, D. Luo, H.G. Craighead. 2005. Single-molecule mobility and spectral measurements in submicrometer fluidic channels J. Appl. Phys. 98, 044903.
• Li, Y., Y. Cu and D. Luo. 2005. DNA fluorescence nanobarcodes for multiplexed pathogen detection, Nature Biotechnology 23, 885-889.
• Gemeinhart, R.A., D. Luo, and W. M. Saltzman. 2005. Cellular fate of a modular DNA delivery system mediated by silica nanoparticles, Biotechnology Progress, 21, 532-537.
• Moreau, L., P. Barthelemy, Y. Li, D. Luo, and M.W. Grinstaff. 2005. Nucleoside Phosphocholine Amphiphile for in vitro DNA transfection, Molecular BioSystems 1 (3), 260-4.
• Freedman, K.O., J. Lee, Y. Li, D. Luo, V.B. Skobeleva, and P.C. Ke. 2005. Diffusion of Single Star-Branched Dendrimer-Like DNA, J. of Phys. Chem. B. 109, 9839-9842.
• Stavis, S.M., J.B. Edel, Y. Li, K.T. Samiee, D Luo and H.G. Craighead. 2005. Detection and identification of nucleic acid engineered fluorescent labels in submicrometer fluidic channels, Nanotechnology, 16, s314-s323.

Progress 01/01/04 to 12/31/04

Outputs
This is a new-investigator grant with a 5-year duration. In the past year we have successfully demonstrated the idea of using DNA as generic instead of genetic materials. In other words, DNA molecules will serve not only as the pay-load (genes), but also as a vector to carry both genes (DNA) and anti-genes (RNA). Specifically, we have constructed, for the first time in the world, branched, Tree-shaped DNA molecules as new DNA materials. The tree-shaped DNA was employed to construct, again for the first time in the world, DNA nanobarcodes that can be used to detect multiple molecular targets simultaneously. Solid phase synthesis schemes and purification processes have been established. These DNA-based structures will serve as scaffoldings to carry both genes and anti-genes (siRNA-producing plasmid) for controlled drug delivery. In addition, insights and knowledge obtained from nucleic acid engineered materials will be used to construct novel RNA building blocks; a hybrid DNA-RNA delivery system is envisioned for the near future.

Impacts
In addition to the last year's expected impact, we have been continuously supporting a local company that is founded by the technology based on our research. New jobs are and will be created for this Ithaca-based, high tech company.

Publications

• Gemeinhart, R.A., D. Luo, and W. M. Saltzman. 2005. Cellular fate of a modular DNA delivery system mediated by silica nanoparticles, Biotechnology Progress, in press.
• Prata, C.A.H., Y. Zhao, P. Barthelemy, Y. Li, D. Luo, T.J. McIntosh, S.J. Lee, and M.W. Grinstaff. 2004. Charge-reversal amphiphiles for gene delivery. J Am Chem Soc 126(39): 12196-7
• Li, Y., Y.D. Tseng, S.Y. Kown, L. dEspaux, J.S. Bunch, P.L McEuen and D. Luo. 2004. Controlled assembly of dendrimer-like DNA. Nature Materials, 3, 38-42.
• Luo, D., Ernest Han, Nadya Belcheva and W. Mark Saltzman. 2004. A self-assembled, modular DNA delivery system mediated by silica nanoparticles J. of Control. Release. 95, 333-341.
• Luo, D. 2004. A new solution to improved gene therapy. Trends in Biotechnology. 22, 101-103.

Progress 01/01/03 to 12/31/03

Outputs
This is a new-investigator grant with a 5-year duration. In this second year of the grant, we have laid the foundations for utilizing DNA as generic instead of genetic materials. In other words, DNA molecules will serve not only as the pay-load (genes), but also as a vector to carry both genes (DNA) and anti-genes (RNA). Specifically, we have constructed, for the first time in the world, branched, Y-shaped DNA molecules as additional building blocks. The Y-shaped DNA was employed to construct, again for the first time in the world, dendrimer-like DNA molecules. Other novel shaped-DNA, including X-, T-, dumb-bell, honey-comb, etc. have also been fabricated in our group. Solid phase synthesis schemes and purification processes are being developed. In addition, a Matlab-based software package has been developed that can calculate and simulate the formation of Y-DNA. The achievement so far is notable. Part of the results will be published in Nature Materials. These DNA-based structures will serve as scaffoldings to carry both genes and anti-genes (siRNA-producing plasmid) for controlled drug delivery. In addition, insights and knowledge obtained from nucleic acid engineered materials will be used to construct novel RNA building blocks; a hybrid DNA-RNA delivery system is envisioned for the near future. A total of 6 papers have been published since 2001. Three of them were published in highly reputable journals including Nature Materials (the best journal in materials science), Macromolecules (the highest ranked journal in polymer science) and Journal of Controlled Release (the highest ranked journal in drug delivery field).

Impacts
Our research on Nucleic Acid Engineering and Nucleic Acid Engineered materials is starting to gain a reputation worldwide. The PI has been invited numerous times to give talks at international conferences. New collaborations have been setup between the PI and the scientists both in US and in Singapore and China. One particular joint project is to use the proposed controlled delivery systems to deliver important genes to agriculturally important animals.

Publications

• No publications reported this period

Progress 01/01/02 to 12/31/02

Outputs
This is a new-investigator grant with a 5-year duration. In this first year of the grant, the PI has established a molecular bioengineering laboratory that focuses on Nucleic Acid Engineering, including RNA engineering and RNA delivery. Since this is also the first year for the PI as a new assistant professor, it is too soon to report detailed accomplishments in the proposed RNA research. However, great progress has been made in setting up the laboratory, preparing RNA --especially small inhibitory RNA (lamin A/C gene has been cloned into an siRNA expression vector, pSilencer), and training both graduate and undergraduate students. A novel delivery vector that is based on unique and unusual DNA nanostructures has also been constructed. This vector is capable of conjugating multiple RNA molecules as well as DNA molecules, making it an ideal modular multi-drug carrier for delivery. The achievement of this vector was reported in a platform presentation at the International Conferences on Nano and Micro Systems held in Aug. 2002. An invention disclosure was submitted to the Cornell University Research Foundation, and a patent application is being drafted. In addition, a paper titled AControlled assembly of dendrimer-like DNA nanostructure@ (authors: Y. Li, Y. Tseng, L. D=espaux and D. Luo) has been submitted. We will continue our effort in devising controlled gene (as well as anti-gene) delivery systems.

Impacts
We believe that this nucleic acid based, enhanced, modular multi-drug delivery system will provide a universal, flexible and local RNA including siRNA delivery platform. It will be extremely desirable in treating, preventing, and controlling disease that will almost certainly improve human and animal health in future.

Publications

• No publications reported this period