The goal of this program is to advance the current compacted DNA nanoparticle based gene therapy technology to enable efficient and long-lasting gene delivery to dividing and non-dividing cells. The program will merge experts with molecular bioengineering, physics, chemistry, and computer science backgrounds at OUHSC, Stanford University and Copernicus Therapeutics, Inc, to accelerate essential preclinical steps for effective non-viral gene therapy. The plan is to engineer DNA vectors with efficient uptake and transport through the plasma membrane that can provide persistent transgene expression without toxicity. This technology can unimolecularly compact DNA with lysine polymers substituted with polyethylene glycol (PEG) into neutral charge nanoparticles with radii of less than 18 nm. These particles can penetrate the cell membrane via nucleolin receptor associated endocytosis and cross the nuclear membrane pore to the nucleus within 15 minutes. The DNA condensation formulation will compact either linear or circular DNA enabling us to eliminate plasmid backbone sequences known to play a significant role in inhibiting gene expression. The potential scientific and clinical benefits of these enhancements are substantial. While our ultimate aim is to use gene transfer to treat human ocular disease, we plan to address basic biological questions that will be important for rational design of vectors for gene therapy applications. Given the dangers inherent in the use of viral vectors, our strategy will enable us to access the favorable aspects of viral vectors while providing the safety and pharmaceutical qualities inherent in non-viral gene delivery systems. Towards this goal, we are working on developing new non-viral vectors for gene transfer to ocular tissues and establishing the cellular and molecular mechanisms involved in gene transduction.
Three aims are proposed to optimize, mechanistically assess, and test our nanoparticle technology.
Aim 1 will generate and compare the efficiency and longevity of EGFP expression between standard circular plasmid vectors and linear or minicircle constructs lacking the vector backbone sequence.
The aim will also combine two novel gene therapy technologies, compacted DNA nanoparticles and pEPI-1 vector containing S/MAR sequence to develop an efficient and persistent gene transfer strategy in vivo. The effect of different vector sequences on promoter specificity will be assessed with two commonly used promoters in retinal gene therapy trials. To direct specific rod photoreceptor expression we will use the mouse opsin promoter (MOP) and to direct expression in the retinal pigment epithelium, we will use the vitelliform macular dystrophy 2 (VMD2) promoter. The constructs will be compacted and subretinally injected into WT mice during development at postnatal day 5 (P5) and in adults (P30). Injections at P5 will evaluate the efficacy of the nanoparticles in transfecting dividing retinal progenitor cells, and results will be relevant for the treatment of early onset eye diseases. Injections in adults will evaluate the efficacy of the nanoparticles in post-mitotic cells which is an appropriate experimental paradigm for treating late onset ocular diseases.
Aim 2 will assess potential barriers to clinical vector application by evaluating particles uptake, trafficking, mechanisms of vector silencing, and in vivo safety.
Aim 3 will test the efficacy of the vectors in rescuing the phenotypes in two well-known disease models: RPE65-/- (Leber's congenital amaurosis) and ABCR-/- (Stargardt's macular dystrophy).

Public Health Relevance

This program is designed to advance our compacted DNA nanotechnology to facilitate its future use as a clinical gene therapy mechanism for ocular diseases. We will assess and optimize cell uptake, intracellular stability (by bypassing the cell lysosomal degradation machinery), trafficking to the nucleus, and mechanisms of silencing in order to achieve high levels and persistent transgene expression without toxicity. Our compaction procedure can condense a single DNA molecule in the presence of multivalent cations and acetate as a counterion into highly ordered rod structure that is less than 12 nm in diameter. We plan on testing the efficiency of this technology in ocular delivery.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY018656-05
Application #
8204931
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Shen, Grace L
Project Start
2008-01-01
Project End
2013-06-30
Budget Start
2012-01-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$351,600
Indirect Cost
$111,600
Name
University of Oklahoma Health Sciences Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
878648294
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
Stuck, Michael W; Conley, Shannon M; Shaw, Ryan A et al. (2014) Electrophysiological characterization of rod and cone responses in the baboon nonhuman primate model. Adv Exp Med Biol 801:67-73
Mitra, Rajendra N; Han, Zongchao; Merwin, Miles et al. (2014) Synthesis and characterization of glycol chitosan DNA nanoparticles for retinal gene delivery. ChemMedChem 9:189-96
Mitra, Rajendra N; Merwin, Miles J; Han, Zongchao et al. (2014) Yttrium oxide nanoparticles prevent photoreceptor death in a light-damage model of retinal degeneration. Free Radic Biol Med 75:140-8
Chakraborty, Dibyendu; Rodgers, Karla K; Conley, Shannon M et al. (2013) Structural characterization of the second intra-discal loop of the photoreceptor tetraspanin RDS. FEBS J 280:127-38
Koirala, Adarsha; Conley, Shannon M; Naash, Muna I (2013) A review of therapeutic prospects of non-viral gene therapy in the retinal pigment epithelium. Biomaterials 34:7158-67
Han, Zongchao; Guo, Junjing; Conley, Shannon M et al. (2013) Retinal angiogenesis in the Ins2(Akita) mouse model of diabetic retinopathy. Invest Ophthalmol Vis Sci 54:574-84
Koirala, Adarsha; Conley, Shannon M; Makkia, Rasha et al. (2013) Persistence of non-viral vector mediated RPE65 expression: case for viability as a gene transfer therapy for RPE-based diseases. J Control Release 172:745-52
Koirala, Adarsha; Makkia, Rasha S; Conley, Shannon M et al. (2013) S/MAR-containing DNA nanoparticles promote persistent RPE gene expression and improvement in RPE65-associated LCA. Hum Mol Genet 22:1632-42
Stuck, Michael W; Conley, Shannon M; Naash, Muna I (2012) Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina. PLoS One 7:e32484
Han, Zongchao; Koirala, Adarsha; Makkia, Rasha et al. (2012) Direct gene transfer with compacted DNA nanoparticles in retinal pigment epithelial cells: expression, repeat delivery and lack of toxicity. Nanomedicine (Lond) 7:521-39

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