A small molecule ApoE4-targeted therapeutic candidate that normalizes sirtuin 1 levels and improves cognition in an Alzheimer's disease mouse model.

We describe here the results from the testing of a small molecule first-in-class apolipoprotein E4 (ApoE4)-targeted sirtuin1 (SirT1) enhancer, A03, that increases the levels of the neuroprotective enzyme SirT1 while not affecting levels of neurotoxic sirtuin 2 (SirT2) in vitro in ApoE4-transfected cells. A03 was identified by high-throughput screening (HTS) and found to be orally bioavailable and brain penetrant. In vivo, A03 treatment increased SirT1 levels in the hippocampus of 5XFAD-ApoE4 (E4FAD) Alzheimer's disease (AD) model mice and elicited cognitive improvement while inducing no observed toxicity. We were able to resolve the enantiomers of A03 and show using in vitro models that the L-enantiomer was more potent than the corresponding D-enantiomer in increasing SirT1 levels. ApoE4 expression has been shown to decrease the level of the NAD-dependent deacetylase and major longevity determinant SirT1 in brain tissue and serum of AD patients as compared to normal controls. A deficiency in SirT1 level has been recently implicated in increased tau acetylation, a dominant post-translational modification and key pathological event in AD and tauopathies. Therefore, as a novel approach to therapeutic development for AD, we targeted identification of compounds that enhance and normalize brain SirT1 levels.

Chronic nicotine improves cognitive and social impairment in mice overexpressing wild type α-synuclein.

In addition to dopaminergic and motor deficits, patients with Parkinson's disease (PD) suffer from non-motor symptoms, including early cognitive and social impairment, that do not respond well to dopaminergic therapy. Cholinergic deficits may contribute to these problems, but cholinesterase inhibitors have limited efficacy. Mice over-expressing α-synuclein, a protein critically associated with PD, show deficits in cognitive and social interaction tests, as well as a decrease in cortical acetylcholine. We have evaluated the effects of chronic administration of nicotine in mice over-expressing wild type human α-synuclein under the Thy1-promoter (Thy1-aSyn mice). Nicotine was administered subcutaneously by osmotic minipump for 6 months from 2 to 8 months of age at 0.4 mg/kg/h and 2.0 mg/kg/h. The higher dose was toxic in the Thy1-aSyn mice, but the low dose was well tolerated and both doses ameliorated cognitive impairment in Y-maze performance after 5 months of treatment. In a separate cohort of Thy1-aSyn mice, nicotine was administered at the lower dose for one month beginning at 5 months of age. This treatment partially eliminated the cognitive deficit in novel object recognition and social impairment. In contrast, chronic nicotine did not improve motor deficits after 2, 4 or 6 months of treatment, nor modified α-synuclein aggregation, tyrosine hydroxylase immunostaining, synaptic and dendritic markers, or microglial activation in Thy1-aSyn mice. These results suggest that cognitive and social impairment in synucleinopathies like PD may result from deficits in cholinergic neurotransmission and may benefit from chronic administration of nicotinic agonists.

Human Neural Stem Cell Transplantation Rescues Functional Deficits in R6/2 and Q140 Huntington's Disease Mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder with no disease-modifying treatment. Expansion of the glutamine-encoding repeat in the Huntingtin (HTT) gene causes broad effects that are a challenge for single treatment strategies. Strategies based on human stem cells offer a promising option. We evaluated efficacy of transplanting a good manufacturing practice (GMP)-grade human embryonic stem cell-derived neural stem cell (hNSC) line into striatum of HD modeled mice. In HD fragment model R6/2 mice, transplants improve motor deficits, rescue synaptic alterations, and are contacted by nerve terminals from mouse cells. Furthermore, implanted hNSCs are electrophysiologically active. hNSCs also improved motor and late-stage cognitive impairment in a second HD model, Q140 knockin mice. Disease-modifying activity is suggested by the reduction of aberrant accumulation of mutant HTT protein and expression of brain-derived neurotrophic factor (BDNF) in both models. These findings hold promise for future development of stem cell-based therapies.

Improvement of neuropathology and transcriptional deficits in CAG 140 knock-in mice supports a beneficial effect of dietary curcumin in Huntington's disease.

BACKGROUND: No disease modifying treatment currently exists for Huntington's disease (HD), a fatal neurodegenerative disorder characterized by the formation of amyloid-like aggregates of the mutated huntingtin protein. Curcumin is a naturally occurring polyphenolic compound with Congo red-like amyloid binding properties and the ability to cross the blood brain barrier. CAG140 mice, a knock-in (KI) mouse model of HD, display abnormal aggregates of mutant huntingtin and striatal transcriptional deficits, as well as early motor, cognitive and affective abnormalities, many months prior to exhibiting spontaneous gait deficits, decreased striatal volume, and neuronal loss. We have examined the ability of life-long dietary curcumin to improve the early pathological phenotype of CAG140 mice. RESULTS: KI mice fed a curcumin-containing diet since conception showed decreased huntingtin aggregates and increased striatal DARPP-32 and D1 receptor mRNAs, as well as an amelioration of rearing deficits. However, similar to other antioxidants, curcumin impaired rotarod behavior in both WT and KI mice and climbing in WT mice. These behavioral effects were also noted in WT C57Bl/6 J mice exposed to the same curcumin regime as adults. However, neither locomotor function, behavioral despair, muscle strength or food utilization were affected by curcumin in this latter study. The clinical significance of curcumin's impairment of motor performance in mice remains unclear because curcumin has an excellent blood chemistry and adverse event safety profile, even in the elderly and in patients with Alzheimer's disease. CONCLUSION: Together with this clinical experience, the improvement in several transgene-dependent parameters by curcumin in our study supports a net beneficial effect of dietary curcumin in HD.

Intravenous nonviral gene therapy causes normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism.

Brain gene-targeting technology is used to reversibly normalize tyrosine hydroxylase (TH) activity in the striatum of adult rats, using the experimental 6-hydroxydopamine model of Parkinson's disease. The TH expression plasmid is encapsulated inside an 85-nm PEGylated immunoliposome (PIL) that is targeted with either the OX26 murine monoclonal antibody (MAb) to the rat transferrin receptor (TfR) or with the mouse IgG2a isotype control antibody. TfRMAb-PIL, or mIgG2a-PIL, is injected intravenously at a dose of 10 microg of plasmid DNA per rat. TfRMAb-PIL, but not mIgG2a-PIL, enters the brain via the transvascular route. The targeting TfRMAb enables the nanocontainer carrying the gene to undergo both receptor-mediated transcytosis across the blood-brain barrier (BBB) and receptor-mediated endocytosis into neurons behind the BBB by accessing the TfR. With this approach, the striatal TH activity ipsilateral to the intracerebral injection of the neurotoxin was normalized and increased from 738 +/- 179 to 5486 +/- 899 pmol/hr per milligram of protein. The TH enzyme activity measurements were corroborated by TH immunocytochemistry, which showed that the entire striatum was immunoreactive for TH after intravenous gene therapy. The normalization of striatal biochemistry was associated with a reversal of apomorphine-induced rotation behavior. Lesioned animals treated with the apomorphine exhibited 20 +/- 5 and 6 +/- 2 rotations/min, respectively, after intravenous administration of the TH plasmid encapsulated in mIgG2a-PIL and TfRMAb-PIL. These studies demonstrate that it is possible to normalize brain enzyme activity by intravenous administration and nonviral gene transfer.

Widespread expression of an exogenous gene in the eye after intravenous administration.

PURPOSE: Gene-targeting technology and tissue-specific gene promoters were used to produce widespread expression of an exogenous gene throughout the eye, including the retinal pigmented epithelium, after noninvasive intravenous administration of a nonviral plasmid formulation. METHODS: An expression plasmid encoding bacterial beta-galactosidase, under the influence of either the simian virus (SV)40 promoter or the glial fibrillary acidic protein (GFAP) gene promoter, was packaged in the interior of 85-nm pegylated immunoliposomes (PIL) targeted to transferrin receptor (TfR)-rich structures with the rat 8D3 monoclonal antibody (mAb) to the mouse TfR. Plasmid DNA was packaged in the 8D3-PIL and injected intravenously into adult female BALB/c mice at a dose of 5 micro g DNA per mouse. The eyes were removed 48 hours later, and frozen sections were prepared for beta-galactosidase histochemistry and rhodopsin, TfR, or GFAP immunocytochemistry. RESULTS: There was diffuse expression of the SV40/beta-galactosidase gene in the retinal pigmented epithelium. The cellular specificity of gene expression was influenced by the promoter used in the gene construct, evidenced by the fact that gene expression in the inner retina was induced with the GFAP promoter. The beta-galactosidase gene was also widely expressed in the TfR-rich epithelial structures of the eye, including the ciliary body, the iris, the sebaceous glands of the tarsal plate, and the epithelium of the cornea. When the anti-TfR mAb on the PIL was replaced with the rat IgG isotype control, there was no gene expression in the eye. CONCLUSIONS: Widespread expression of an exogenous gene throughout the retina pigmented epithelium or other structures of the eye is possible with a noninvasive intravenous administration of a nonviral plasmid that is reformulated with gene-targeting technology. The cellular specificity of gene expression in the eye can be regulated with the use of cell-specific gene promoters.

Antisense gene therapy of brain cancer with an artificial virus gene delivery system.

Therapeutic genes are delivered to the nuclear compartment of cancer cells following intravenous administration with a non-immunogenic "artificial virus" gene delivery system that uses receptor-specific monoclonal antibodies (MAb) to navigate the biological barriers between the blood and the nucleus of the cancer cell. Mice implanted with intracranial U87 human glial brain tumors are treated with a nonviral expression plasmid encoding antisense mRNA against the human epidermal growth factor receptor gene (EGFR). The plasmid DNA is packaged within the interior of polyethylene glycol-modified (PEGylated) immunoliposomes, and delivered to the brain tumor with MAbs that target the mouse transferrin receptor (TRFR) and the human insulin receptor (INSR). The mouse TRFR MAb enables transport across the tumor vasculature, which is of mouse brain origin, and the INSR MAb causes transport across the plasma membrane and the nuclear membrane of the human brain cancer cell. The lifespan of the mice treated weekly with an intravenous administration of the EGFR antisense gene therapy packaged within the artificial virus is increased 100% relative to mice treated either with a luciferase gene or with saline.