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Koirala A.,The University of Oklahoma Health Sciences Center | Makkia R.S.,The University of Oklahoma Health Sciences Center | Cooper M.J.,Copernicus Therapeutics Inc. | Naash M.I.,The University of Oklahoma Health Sciences Center
Biomaterials | Year: 2011

Previously, we demonstrated that CK30PEG10k-compacted DNA nanoparticles (NPs) efficiently target photoreceptor cells and improve visual function in a retinitis pigmentosa model. Here, we test the ability of these NPs in driving transgene expression in the retinal pigment epithelium (RPE), using an RPE-specific reporter vector (VMD2-eGFP). NPs, uncompacted plasmid, or saline were subretinally delivered to adult BALB/c mice. NP-based expression was specific to RPE cells and caused no deleterious effects on retinal structure and function. eGFP expression levels in NP-injected eyes peaked at post-injection day 2 (PI-2), stabilized at levels ∼3-fold higher than in naked DNA-injected eyes, and remained elevated at the latest time-point examined (PI-30). Unlike naked DNA, which only transfected cells at the site of injection, NPs were able to transfect cells throughout the RPE. Subretinal injections of rhodamine labeled NPs and naked DNA showed comparable initial uptake into RPE cells. However, at PI-7 and -30 days significantly more fluorescence was detected inside the RPE of NP-injected eyes compared to naked DNA, suggesting NPs are stable inside the cell which could possibly lead to higher and sustained expression. Overall, our results demonstrate that NPs can efficiently deliver genes to the RPE and hold great potential for the treatment of RPE-associated diseases. © 2011. Source

Han Z.,The University of Oklahoma Health Sciences Center | Conley S.M.,The University of Oklahoma Health Sciences Center | Makkia R.S.,The University of Oklahoma Health Sciences Center | Cooper M.J.,Copernicus Therapeutics Inc. | Naash M.I.,The University of Oklahoma Health Sciences Center
Journal of Clinical Investigation | Year: 2012

Mutations in the photoreceptor-specific flippase ABCA4 are associated with Stargardt disease and many other forms of retinal degeneration that currently lack curative therapies. Gene replacement is a logical strategy for ABCA4-associated disease, particularly given the current success of traditional viral-mediated gene delivery, such as with adeno-associated viral (AAV) vectors. However, the large size of the ABCA4 cDNA (6.8 kbp) has hampered progress in the development of genetic treatments. Nonviral DNA nanoparticles (NPs) can accommodate large genes, unlike traditional viral vectors, which have capacity limitations. We utilized an optimized DNA NP technology to subretinally deliver ABCA4 to Abca4-deficient mice. We detected persistent ABCA4 transgene expression for up to 8 months after injection and found marked correction of functional and structural Stargardt phenotypes, such as improved recovery of dark adaptation and reduced lipofuscin granules. These data suggest that DNA NPs may be an excellent, clinically relevant gene delivery approach for genes too large for traditional viral vectors. Source

Boylan N.J.,Johns Hopkins University | Kim A.J.,Johns Hopkins University | Kim A.J.,Wilmer Eye Institute | Suk J.S.,Johns Hopkins University | And 8 more authors.
Biomaterials | Year: 2012

Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-l-lysine (PEG-CK 30), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-l-histidine was inserted between PEG and poly-l-lysine to form a triblock copolymer system, PEG-CH 12K 18. Inclusion of poly-l-histidine increased the buffering capacity of PEG-CH 12K 18 to levels comparable with branched polyethyleneimine. PEG-CH 12K 18 compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK 30 DNA nanoparticles. PEG-CH 12K 18 DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH 12K 18 DNA nanoparticles improved the invitro gene transfer by ∼20-fold over PEG-CK 30 DNA nanoparticles, and invivo gene transfer to lung airways in BALB/c mice by~3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles. © 2011 Elsevier Ltd. Source

Koirala A.,The University of Oklahoma Health Sciences Center | Makkia R.S.,The University of Oklahoma Health Sciences Center | Conley S.M.,The University of Oklahoma Health Sciences Center | Cooper M.J.,Copernicus Therapeutics Inc. | Naash M.I.,The University of Oklahoma Health Sciences Center
Human Molecular Genetics | Year: 2013

Mutations in genes in the retinal pigment epithelium (RPE) cause or contribute to debilitating ocular diseases, including Leber's congenital amaurosis (LCA). Genetic therapies, particularly adeno-associated viruses (AAVs), are a popular choice for monogenic diseases; however, the limited payload capacity of AAVs combined with the large number of retinal disease genes exceeding that capacity make the development of alternative delivery methods critical. Here, we test the ability of compacted DNA nanoparticles (NPs) containing a plasmid with a scaffold matrix attachment region (S/MAR) and vitelliform macular dystrophy 2 (VMD2) promoter to target the RPE, drive long-term, tissue-specific gene expression and mediate proof-of-principle rescue in the rpe65-/- model of LCA. We show that the S/MAR-containing plasmid exhibited reporter gene expression levels several fold higher than plasmid or NPs without S/MARs. Importantly, this expression was highly persistent, lasting up to 2 years (last timepoint studied). We therefore selected this plasmid for testing in the rpe65-/- mouse model and observe that NP or plasmid VMD2-hRPE65-S/MAR led to structural and functional improvements in the LCA disease phenotype. These results indicate that the non-viral delivery of hRPE65 vectors can result in persistent, therapeutically efficacious gene expression in the RPE. © The Author 2013. Published by Oxford University Press. All rights reserved. Source

Han Z.,The University of Oklahoma Health Sciences Center | Han Z.,University of North Carolina at Chapel Hill | Banworth M.J.,The University of Oklahoma Health Sciences Center | Makkia R.,The University of Oklahoma Health Sciences Center | And 4 more authors.
FASEB Journal | Year: 2015

Mutations in the rhodopsin gene cause retinal degeneration and clinical phenotypes including retinitis pigmentosa (RP) and congenital stationary night blindness. Effective gene therapies have been difficult to develop, however, because generating precise levels of rhodopsin expression is critical; overexpression causes toxicity, and underexpression would result in incomplete rescue. Current gene delivery strategies routinely use cDNA-based vectors for gene targeting; however, inclusion of noncoding components of genomic DNA (gDNA) such as intronsmay help promotemore endogenous regulation of gene expression. Here we test the hypothesis that inclusion of genomic sequences from the rhodopsin gene can improve the efficacy of rhodopsin gene therapy in the rhodopsin knockout (RKO) mouse model of RP. We utilize our compacted DNA nanoparticles (NPs), which have the ability to transfer larger and more complex genetic constructs, to deliver murine rhodopsin cDNA or gDNA. We show functional and structural improvements in RKO eyes for up to 8 months after NP-mediated gDNA but not cDNA delivery. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at time pointswhen cones in the RKO model are degenerated. These results suggest that inclusion of native expression elements, such as introns, can significantly enhance gene expression and therapeutic efficacy and may become an essential option in the array of available gene delivery tools. © FASEB. Source

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