Cleveland, OH, United States
Cleveland, OH, United States

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Harmon B.T.,Northeastern University | Aly A.E.,Copernicus Therapeutics Inc. | Padegimas L.,Northeastern University | Sesenoglu-Laird O.,Northeastern University | And 2 more authors.
Gene Therapy | Year: 2014

Viral vectors are a commonly used method for gene therapy because of their highly efficient transduction of cells. However, many vectors have a small genetic capacity, and their potential for immunogenicity can limit their usefulness. Moreover, for disorders of the central nervous system (CNS), the need for invasive surgical delivery of viruses to the brain also detracts from their clinical applicability. Here, we show that intranasal delivery of unimolecularly compacted DNA nanoparticles (DNA NPs), which consist of single molecules of plasmid DNA encoding enhanced green fluorescent protein (eGFP) compacted with 10 kDa polyethylene glycol (PEG)-substituted lysine 30-Mers (CK30PEG10k), successfully transfect cells in the rat brain. Direct eGFP fluorescence microscopy, eGFP-immunohistochemistry (IHC) and eGFP-ELISA all demonstrated eGFP protein expression 2 days after intranasal delivery. eGFP-positive cells were found throughout the rostral-caudal axis of the brain, most often adjacent to capillary endothelial cells. This localization provides evidence for distribution of the nasally administered DNA NPs via perivascular flow. These results are the first report that intranasal delivery of DNA NPs can bypass the blood-brain barrier and transfect and express the encoded protein in the rat brain, affording a non-invasive approach for gene therapy of CNS disorders. © 2014 Macmillan Publishers Limited.


Cai X.,The University of Oklahoma Health Sciences Center | Conley S.M.,The University of Oklahoma Health Sciences Center | Nash Z.,The University of Oklahoma Health Sciences Center | Fliesler S.J.,Research Service | And 3 more authors.
FASEB Journal | Year: 2010

The purpose of the present study was to test the therapeutic efficiency and safety of compacted-DNA nanoparticle-mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild-type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow (rds+/-) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI-2 to PI-7) for P5 injections, stabilized at levels 2-fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI-120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle-injected eyes reached wild-type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI-120 revealed significant improvement in outer segment structures in P5 nanoparticle-injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL-6 or TNF-α mRNA in P5 or P22 nanoparticle-dosed eyes at either PI-2 or PI-30. Thus, compacted-DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle-based gene replacement therapy for treatment of human retinal degenerations. © FASEB.


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.


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.


Han Z.,The University of Oklahoma Health Sciences Center | Koirala A.,The University of Oklahoma Health Sciences Center | Makkia R.,The University of Oklahoma Health Sciences Center | Cooper M.J.,Copernicus Therapeutics Inc. | Naash M.I.,The University of Oklahoma Health Sciences Center
Nanomedicine | Year: 2012

Aim: To evaluate the safety of compacted DNA nanoparticles (NPs) in retinal pigment epithelial (RPE) cells. Materials & methods: Enhanced GFP expression cassettes controlled by the RPE-specific vitelloform macular dystrophy promoter were constructed with and without a bacterial backbone and compacted into NPs formulated with polyethylene glycol-substituted lysine 30-mers. Single or double subretinal injections were administered in adult BALB/c mice. Expression levels of enhanced GFP, proinflammatory cytokines and neutrophil/macrophage mediators, and retinal function by electroretinogram were evaluated at different time-points postinjection. Results: Immunohistochemistry and real-time PCR demonstrated that NPs specifically transfect RPE cells at a higher efficiency than naked DNA and similar results were observed after the second injection. At 6 h postinjections, a transient inflammatory response was observed in all cohorts, including saline, indicating an adverse effect to the injection procedure. Subsequently, no inflammation was detected in all experimental groups. Conclusion: This study demonstrates the safety and efficacy of NP-mediated RPE gene transfer therapy following multiple subretinal administrations. © 2012 Future Medicine Ltd.


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.


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.


Han Z.,The University of Oklahoma Health Sciences Center | Conley S.M.,The University of Oklahoma Health Sciences Center | Makkia R.,The University of Oklahoma Health Sciences Center | Guo J.,The University of Oklahoma Health Sciences Center | And 2 more authors.
PLoS ONE | Year: 2012

Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors. © 2012 Han et al.


Padegimas L.,Copernicus Therapeutics Inc. | Kowalczyk T.H.,Copernicus Therapeutics Inc. | Adams S.,Copernicus Therapeutics Inc. | Gedeon C.R.,Copernicus Therapeutics Inc. | And 7 more authors.
Molecular Therapy | Year: 2012

Efficient and prolonged human cystic fibrosis transmembrane conductance regulator (hCFTR) expression is a major goal for cystic fibrosis (CF) lung therapy. A hCFTR expression plasmid was optimized as a payload for compacted DNA nanoparticles formulated with polyethylene glycol (PEG)-substituted 30-mer lysine peptides. A codon-optimized and CpG-reduced hCFTR synthetic gene (CO-CFTR) was placed in a polyubiquitin C expression plasmid. Compared to hCFTR complementary DNA (cDNA), CO-CFTR produced a ninefold increased level of hCFTR protein in transfected HEK293 cells and, when compacted as DNA nanoparticles, produced a similar improvement in lung mRNA expression in Balb/c and fatty acid binding protein promoter (FABP) CF mice, although expression duration was transient. Various vector modifications were tested to extend duration of CO-CFTR expression. A novel prolonged expression (PE) element derived from the bovine growth hormone (BGH) gene 3′ flanking sequence produced prolonged expression of CO-CFTR mRNA at biologically relevant levels. A time course study in the mouse lung revealed that CO-CFTR mRNA did not change significantly, with CO-CFTR/mCFTR geometric mean ratios of 94% on day 2, 71% on day 14, 53% on day 30, and 14% on day 59. Prolonged CO-CFTR expression is dependent on the orientation of the PE element and its transcription, is not specific to the UbC promoter, and is less dependent on other vector backbone elements. © The American Society of Gene & Cell Therapy.


Patent
Copernicus Therapeutics Inc. | Date: 2011-01-19

A method of compacting a nucleic acid molecule comprisingadding one or more nucleic acid molecules to a solution comprising polycations, whereby compacted particles of DNA and polycation are formed which are unimolecular with respect to nucleic acid.

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