Systemic delivery of mutant huntingtin lowering antisense oligonucleotides to the brain using apolipoprotein A-I nanodisks for Huntington disease.

Efficient delivery of therapeutics to the central nervous system (CNS) remains a major challenge for the treatment of neurological diseases. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion mutation in the HTT gene which codes for a toxic mutant huntingtin (mHTT) protein. Pharmacological reduction of mHTT in the CNS using antisense oligonucleotides (ASO) ameliorates HD-like phenotypes in rodent models of HD, with such therapies being investigated in clinical trials for HD. In this study, we report the optimization of apolipoprotein A-I nanodisks (apoA-I NDs) as vehicles for delivery of a HTT-targeted ASO (HTT ASO) to the brain and peripheral organs for HD. We demonstrate that apoA-I wild type (WT) and the apoA-I K133C mutant incubated with a synthetic lipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, can self-assemble into monodisperse discoidal particles with diameters <20 nm that transmigrate across an in vitro blood-brain barrier model of HD. We demonstrate that apoA-I NDs are well tolerated in vivo, and that apoA-I K133C NDs show enhanced distribution to the CNS and peripheral organs compared to apoA-I WT NDs following systemic administration. ApoA-I K133C conjugated with HTT ASO forms NDs (HTT ASO NDs) that induce significant mHTT lowering in the liver, skeletal muscle and heart as well as in the brain when delivered intravenously in the BACHD mouse model of HD. Furthermore, HTT ASO NDs increase the magnitude of mHTT lowering in the striatum and cortex compared to HTT ASO alone following intracerebroventricular administration. These findings demonstrate the potential utility of apoA-I NDs as biocompatible vehicles for enhancing delivery of mutant HTT lowering ASOs to the CNS and peripheral organs for HD.

Delivery of mutant huntingtin-lowering antisense oligonucleotides to the brain by intranasally administered apolipoprotein A-I nanodisks.

Lowering mutant huntingtin (mHTT) in the central nervous system (CNS) using antisense oligonucleotides (ASOs) is a promising approach currently being evaluated in clinical trials for Huntington disease (HD). However, the therapeutic potential of ASOs in HD patients is limited by their inability to cross the blood-brain barrier (BBB). In non-human primates, intrathecal infusion of ASOs results in limited brain distribution, with higher ASO concentrations in superficial regions and lower concentrations in deeper regions, such as the basal ganglia. To address the need for improved delivery of ASOs to the brain, we are evaluating the therapeutic potential of apolipoprotein A-I nanodisks (apoA-I NDs) as novel delivery vehicles for mHTT-lowering ASOs to the CNS after intranasal administration. Here, we have demonstrated the ability of apoA-I nanodisks to bypass the BBB after intranasal delivery in the BACHD model of HD. Following intranasal administration of apoA-I NDs, apoA-I protein levels were elevated along the rostral-caudal brain axis, with highest levels in the most rostral brain regions including the olfactory bulb and frontal cortex. Double-label immunohistochemistry indicates that both the apoA-I and ASO deposit in neurons. Most importantly, a single intranasal dose of apoA-I ASO-NDs significantly reduces mHTT levels in the brain regions most affected in HD, namely the cortex and striatum. This approach represents a novel non-invasive means for improving delivery and brain distribution of oligonucleotide therapies and enhancing likelihood of efficacy. Improved ASO delivery to the brain has widespread application for treatment of many other CNS disorders.

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.

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.

Intranasal gene delivery for treating Parkinson's disease: overcoming the blood-brain barrier.

INTRODUCTION: Developing a disease-modifying gene therapy for Parkinson's disease (PD) has been a high priority for over a decade. However, due to the inability of large biomolecules to cross the blood-brain barrier (BBB), the only means of delivery to the brain has been intracerebral infusion. Intranasal administration offers a non-surgical means of bypassing the BBB to deliver neurotrophic factors, and the genes encoding them, directly to the brain. AREAS COVERED: This review summarizes: i) evidence demonstrating intranasal delivery to the brain of a number of biomolecules having therapeutic potential for various CNS disorders; and ii) evidence demonstrating neuroprotective efficacy of a subset of biomolecules specifically for PD. The intersection of these two spheres represents the area of opportunity for development of new intranasal gene therapies for PD. To that end, our laboratory showed that intranasal administration of glial cell line-derived neurotrophic factor (GDNF), or plasmid DNA nanoparticles encoding GDNF, provides neuroprotection in a rat model of PD, and that the cells transfected by the nanoparticle vector are likely to be pericytes. EXPERT OPINION: A number of genes encoding neurotrophic factors have therapeutic potential for PD, but few have been tested by the intranasal route and shown to be neuroprotective in a model of PD. Intranasal delivery provides a largely unexplored, promising approach for development of a non-invasive gene therapy for PD.