Focused ultrasound enhances transgene expression of intranasal hGDNF DNA nanoparticles in the sonicated brain regions.

The therapeutic potential of many gene therapies is limited by their inability to cross the blood brain barrier (BBB). While intranasal administration of plasmid DNA nanoparticles (NPs) offers a non-invasive approach to bypass the BBB, it is not targeted to disease-relevant brain regions. Here, our goal was to determine whether focused ultrasound (FUS) can enrich intranasal delivery of our plasmid DNA NPs to target deeper brain regions, in this case the regions most affected in Parkinson's disease. Combining FUS with intranasal administration resulted in enhanced delivery of DNA NPs to the rodent brain, by recruitment and transfection of microglia. FUS increased transgene expression by over 3-fold after intranasal administration compared to intravenous administration. Additionally, FUS with intranasal delivery increased transgene expression in the sonicated hemisphere by over 80%, altered cellular transfection patterns at the sonication sites, and improved penetration of plasmid NPs into the brain parenchyma (with a 1-fold and 3-fold increase in proximity of transgene expression to neurons in the forebrain and midbrain respectively, and a 40% increase in proximity of transgene expression to dopaminergic neurons in the substantia nigra). These results provide evidence in support of using FUS to improve transgene expression after intranasal delivery of non-viral gene therapies.

Suprachoroidally Delivered DNA Nanoparticles Transfect Retina and Retinal Pigment Epithelium/Choroid in Rabbits.

Purpose: This study evaluated ocular tolerability and transfectability of nonviral DNA nanoparticles (DNPs) after microneedle-based suprachoroidal (SC) administration, in comparison to subretinal (SR) administration. Methods: The DNPs consisted of a single copy of plasmid DNA with a polyubiquitin C/luciferase transcriptional cassette compacted with 10 kDa PEG-substituted lysine 30-mer peptides (CK30PEG10k). New Zealand White rabbits (n = 4 per group) received a unilateral SC injection (0.1 mL via a microneedle technique) of ellipsoid-shaped DNPs, rod-shaped DNPs, or saline (negative control). A cohort of rabbits (n = 4) also received a single unilateral SR injection (0.05 mL via a transvitreal approach) of rod-shaped DNPs. At day 7, luciferase activity was measured in the retina and retinal pigment epithelium (RPE)-choroid via bioluminescence assay. A cohort of rabbits received a SC injection of analogous DNPs to assess spread of DNP injectate in the suprachoroidal space (SCS) via optical coherent tomography and histology. Results: Suprachoroidal injection of DNPs resulted in reversible opening of the SCS circumferentially and posteriorly and was generally well tolerated, with no significant ocular examination score changes, intraocular pressure abnormalities, or changes in electroretinography amplitudes on day 7 compared to the baseline. High luciferase activity was observed in the retina and RPE-choroid of eyes that received SC DNPs (rod and ellipsoid shape) and SR DNPs (rod shape) compared to controls. The mean luciferase activity in RPE-choroid and retina was comparable between SC and SR administrations. Transfection in the RPE-choroid was approximately 10-fold higher than in the retina after either SC or SR administration of DNPs. Conclusions: Suprachoroidal and SR administration of DNPs resulted in comparable transfection of retina and RPE-choroid. Translational Relevance: Suprachoroidal delivery of DNPs offers the potential to precisely target chorioretinal tissues while avoiding surgical risks associated with SR injection, and it may offer an office-based nonsurgical gene therapy option for the treatment of retinal diseases.

Intranasal Delivery of pGDNF DNA Nanoparticles Provides Neuroprotection in the Rat 6-Hydroxydopamine Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) gene therapy could offer a disease-modifying treatment for Parkinson's disease (PD). Here, we report that plasmid DNA nanoparticles (NPs) encoding human GDNF administered intranasally to rats induce transgene expression in the brain and protect dopamine neurons in a model of PD. To first test whether intranasal administration could transfect cells in the brain, rats were sacrificed 1 week after intranasal pGDNF NPs or the naked plasmid. GDNF ELISA revealed significant increases in GDNF expression throughout the brain for both treatments. To assess whether expression was sufficient to protect dopamine neurons, naked pGDNF and pGDNF DNA NPs were given intranasally 1 week before a unilateral 6-hydroxydopamine lesion in a rat model of PD. Three to four weeks after the lesion, amphetamine-induced rotational behavior was reduced, and dopaminergic fiber density and cell counts in the lesioned substantia nigra and nerve terminal density in the lesioned striatum were significantly preserved in rats given intranasal pGDNF. The NPs afforded a greater level of neuroprotection than the naked plasmid. These results provide proof-of-principle that intranasal administration of pGDNF DNA NPs can offer a non-invasive, non-viral gene therapy approach for early-stage PD.

Intranasal delivery of hGDNF plasmid DNA nanoparticles results in long-term and widespread transfection of perivascular cells in rat brain.

The intranasal route of administration allows large therapeutics to circumvent the blood-brain barrier and be delivered directly to the CNS. Here we examined the distribution and pattern of cellular transfection, and the time course of transgene expression, in the rat brain after intranasal delivery of plasmid DNA nanoparticles (NPs) encoding hGDNF fused with eGFP. Intranasal administration of these NPs resulted in transfection and transgene expression throughout the rat brain, as indicated by eGFP ELISA and eGFP-positive cell counts. Most of the transfected cells were abluminal and immediately adjacent to capillaries and are likely pericytes, consistent with their distribution by perivascular transport. Intranasal administration of these plasmid DNA NPs resulted in significant, long-term transgene expression in rat brain, with highest levels at 1 week and continued expression for 6 months. These results provide evidence in support of intranasal DNA NPs as a non-invasive, long-term gene therapy approach for various CNS disorders.

Optimizing Non-viral Gene Therapy Vectors for Delivery to Photoreceptors and Retinal Pigment Epithelial Cells.

Considerable progress has been made in the design and delivery of non-viral gene therapy vectors, but, like their viral counterparts, therapeutic levels of transgenes have not met the requirements for successful clinical applications so far. The biggest advantage of polymer-based nanoparticle vectors is the ease with which they can be modified to increase their ability to penetrate the cell membrane and target specific cells by simply changing the formulation of the nanoparticle compaction. We took advantage of this characteristic to improve transfection rates of our particles to meet the transgene levels which will be needed for future treatment of patients. For this study, we successfully investigated the possibility of our established pegylated polylysine particles to be administered via intravitreal rather than subretinal route to ease the damage during injection. We also demonstrated that our particles are flexible enough to sustain changes in the formulation to accommodate additional targeting sequences without losing their efficiency in transfecting neuronal cells in the retina. Together, these results give us the opportunity to even further improve our particles.

Intracerebral injections of DNA nanoparticles encoding for a therapeutic gene provide partial neuroprotection in an animal model of neurodegeneration.

This study reports proof of concept for administering compacted DNA nanoparticles (DNPs) intracerebrally as a means to protect against neurotoxin-induced neurodegeneration of dopamine (DA) neurons. In this study we used DNPs that encoded for human glial cell line-derived neurotrophic factor (hGDNF); GDNF is a potent neurotrophic factor for DA neurons. Intracerebral injections of DNPs into the striatum and/or substantia nigra were performed 1 week before treatment with a neurotoxin. We observed that the number of surviving DA cells, the density of DA fiber terminals and recovery of motor function were greater in animals injected with GDNF-encoding DNPs than in control animals receiving DNPs encoding for an inert reporter gene. The results of these studies are one of the first to demonstrate that a non-viral, synthetic nanoparticle can be used to deliver therapeutic genes to cells in the brain as a means to protect cells against neurotoxin-induced neurodegeneration.

Optimization of hCFTR lung expression in mice using DNA nanoparticles.

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.