Yong Hong Chen

Molecular signatures of disease brain endothelia provide new sites for CNS-directed enzyme therapy

The brain vasculature forms an immense network such that most neural cells are in contact with a microvessel. Here we tested the hypothesis that endothelia lining these vessels can be harnessed to create a cellular reservoir of enzyme replacement therapy to diseased brain. As a model system, we used mice with central nervous system (CNS) deficits due to lysosomal storage disease (LSD mice). The basic premise of this work is that recombinant enzyme expressed in, and secreted from, the vascular endothelia will be endocytosed by underlying neurons and glia, decreasing neuropathology. We screened a phage library in vivo by panning to identify peptides that bound the vascular endothelia in diseased and wild-type mice. Epitopes binding diseased brain were distinct from those panned from normal brain. Moreover, different epitopes were identified in two distinct LSD disease models, implying a unique vascular signature imparted by the disease state. Presentation of these epitopes on the capsid of adeno-associated virus (AAV) expanded the biodistribution of intravenously injected AAV from predominantly liver to include the CNS. Peripheral injection of the epitope-modified AAVs expressing the enzymes lacking in LSD mice reconstituted enzyme activity throughout the brain and improved disease phenotypes in two distinct disease models (pages 1123–1124).

In vivo SELEX for Identification of Brain-penetrating Aptamers

The physiological barriers of the brain impair drug delivery for treatment of many neurological disorders. One delivery approach that has not been investigated for their ability to penetrate the brain is RNA-based aptamers. These molecules can impart delivery to peripheral tissues and circulating immune cells, where they act as ligand mimics or can be modified to carry payloads. We developed a library of aptamers and an in vivo evolution protocol to determine whether specific aptamers could be identified that would home to the brain after injection into the peripheral vasculature. Unlike biopanning with recombinant bacteriophage libraries, we found that the aptamer library employed here required more than 15 rounds of in vivo selection for convergence to specific sequences. The aptamer species identified through this approach bound to brain capillary endothelia and penetrated into the parenchyma. The methods described may find general utility for targeting various payloads to the brain.

Reinstating Aberrant mTORC1 Activity in Huntington’s Disease Mice Improves Disease Phenotypes

Huntingtons disease (HD) is caused by a polyglutamine tract expansion in huntingtin (HTT). Despite HTTs ubiquitous expression, there is early and robust vulnerability in striatum, the cause of which is poorly understood. Here, we provide evidence that impaired striatal mTORC1 activity underlies varied metabolic and degenerative phenotypes in HD brain and show that introducing the constitutively active form of the mTORC1 regulator, Rheb, into HD mouse brain, alleviates mitochondrial dysfunction, aberrant cholesterol homeostasis, striatal atrophy, impaired dopamine signaling, and increases autophagy. We also find that the expression of Rhes, a striatum-enriched mTOR activator, is reduced in HD patient and mouse brain and that exogenous addition of Rhes alleviates motor deficits and improves brain pathology in HD mice. Our combined work indicates that impaired Rhes/mTORC1 activity in HD brain may underlie the notable striatal susceptibility and thus presents a promising therapeutic target for HD therapy.

Sialic Acid Deposition Impairs the Utility of AAV9, but Not Peptide-modified AAVs for Brain Gene Therapy in a Mouse Model of Lysosomal Storage Disease

Recombinant vector systems have been recently identified that when delivered systemically can transduce neurons, glia, and endothelia in the central nervous system (CNS), providing an opportunity to develop therapies for diseases affecting the brain without performing direct intracranial injections. Vector systems based on adeno-associated virus (AAV) include AAV serotype 9 (AAV9) and AAVs that have been re-engineered at the capsid level for CNS tropism. Here, we performed a head-to-head comparison of AAV9 and a capsid modified AAV for their abilities to rescue CNS and peripheral disease in an animal model of lysosomal storage disease (LSD), the mucopolysacharidoses (MPS) VII mouse. While the peptide-modified AAV reversed cognitive deficits, improved storage burden in the brain, and substantially prolonged survival, we were surprised to find that AAV9 provided no CNS benefit. Additional experiments demonstrated that sialic acid, a known inhibitor of AAV9, is elevated in the CNS of MPS VII mice. These studies highlight how disease manifestations can dramatically impact the known tropism of recombinant vectors, and raise awareness to assuming similar transduction profiles between normal and disease models.

A novel gene delivery method transduces porcine pancreatic duct epithelial cells

Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages—delivery approaches that can be translated to humans.

Targeted Gene Silencing to Induce Permanent Sterility

A non-surgical method to induce sterility would be a useful tool to control feral populations of animals. Our laboratories have experience with approaches aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A combination/modification of these methods may provide a useful framework for the design of approaches that can be used to sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: it needs to target a gene essential for fertility. It must involve a method that can selectively silence the gene of interest. It also needs to deliver the silencing agent via a minimally invasive method. Finally, the silencing effect needs to be sustained for many years, so that expansion of the targeted population can be effectively prevented. In this article, we discuss this subject and provide a succinct account of our previous experience with: (i) molecular reagents able to disrupt reproductive cyclicity when delivered to regions of the brain involved in the control of reproduction and (ii) molecular reagents able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.

Applying Gene Silencing Technology to Contraception

Population control of feral animals is often difficult, as it can be dangerous for the animals, labour intensive and expensive. Therefore, a useful tool for control of animal populations would be a non-surgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to non-invasively sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: the target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article, we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include (i) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction and (ii) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.

Cardiac mTORC1 Dysregulation Impacts Stress Adaptation and Survival in Huntington’s Disease

SUMMARY Huntington’s disease (HD) is a dominantly inherited neurological disorder caused by CAG-repeat expansion in exon 1 of Huntingtin (HTT). But in addition to the neurological disease, mutant HTT (mHTT), which is ubiquitously expressed, impairs other organ systems. Indeed, epidemiological and animal model studies suggest higher incidence of and mortality from heart disease in HD. Here, we show that the protein complex mTORC1 is dysregulated in two HD mouse models through a mechanism that requires intrinsic mHTT expression. Moreover, restoring cardiac mTORC1 activity with constitutively active Rheb prevents mortality and relieves the mHTT-induced block to hypertrophic adaptation to cardiac stress. Finally, we show that chronic mTORC1 dysregulation is due in part to mislocalization of endogenous Rheb. These data provide insight into the increased cardiac-related mortality of HD patients, with cardiac mHTT expression inhibiting mTORC1 activity, limiting heart growth, and decreasing the heart’s ability to compensate to chronic stress.

420. Tropism-Modified Adeno-Associated Virus Vector Mediates Targeting of Brain Vascular Endothelium In Vivo

Top of pageAbstract Adeno-associated virus (AAV) is an attractive vector for gene delivery because of its low toxicity and ability to express transgenes for many months. Collectively, the different serotypes of AAV show broad tissue and cell tropism. However, AAV does not preferentially transduce endothelia when administered in vivo. In our approaches to develop gene therapy for the CNS manifestations of the lysosomal storage diseases (mucopolysaccharidoses; [e.g. Beta-glucuronidase deficiency]) and the ceroid lipofuscinoses (e.g. CLN2 or tripeptidyl protease I [TPP-1] deficiency) endothelia remain an attractive target. Endothelial expression of TPP-1 or beta-glucuronidase results in both apical and basolateral secretion of enzyme; in the brain, this would allow exposure of recombinant enzyme to the underlying brain parenchyma. We used phage display to identify peptides that mediate selective and efficient binding to brain endothelial cells in vivo. Selected peptides were genetically incorporated into the AAV2 capsid, and peptide modified viruses (PM-AAVs) tested in vitro and in vivo. AAV2-ITR genomes were efficiently packaged into PM-AAVs and viral titers were comparable to those obtained with native AAV2 (5-10^11 vector genomes/ml). PM- and native-AAV2 vectors were subsequently injected intravenously via tail vein and vector genomes in various tissues quantified 28 days later. Approximately 10^4 more PM-AAV was present in brain relative to native AAV2. Moreover, while AAV2 genomes were predominantly found in liver, PM-AAV was highest in brain. Our primary data indicate that PM-AAV2 can be retargeted to brain by use of in vivo phage display-derived peptides. This strategy may prove valuable for correction of the CNS deficiencies of several fatal metabolic diseases.

93. Targeting AAV to Brain Vascular Endothelium

Adeno-associated virus (AAV) is an attractive vector for gene delivery because of its low toxicity and ability to express transgenes for many months. Collectively, the different serotypes of AAV show broad tissue and cell tropism. Despite this, AAV does not efficiently transduce endothelia. In our approaches to develop gene therapy for the CNS manifestations of the lysosomal storage diseases (mucopolysaccharidoses; [e.g., beta-glucuronidase deficiency]) and the ceroid lipofuscinoses (e.g., CLN2 or tripeptidyl protease I [TPP-1] deficiency) endothelia remain an attractive target. Endothelial expression of TPP-1 or beta-glucuronidase results in both apical and basolateral secretion of enzyme; in the brain, this would allow exposure of recombinant enzyme to the underlying brain parenchyma. As a first step in developing endothelial-targeted AAV, we panned, in vivo, for brain specific epitopes by phage display. Beta-Glucuronidase deficient and age matched wildtype littermates were used for panning. This was accomplished by canulating the internal carotid artery, injecting phage, and perfusing the mice 5 min later with DMEM. Phage were recovered by homogenizing the brain and incubating the homogenate with bacteria and culture media. After five rounds of panning we isolated a panel of targeting peptides that bound selectively and efficiently to brain endothelia. The motifs isolated from beta-glucuronidase deficient mice were distinct from those panned from heterozygous littermates. Selective peptides have been genetically incorporated into AAV2 VP1 capsid protein at amino acid position 587, a site previously shown to accommodate peptide insertion for directed gene transfer. Recombinant viruses will be assessed for specific delivery to brain relative to AAV2 with native VP1 capsid, in beta-glucuronidase and wildtype mice. Our studies provide possible strategies for correction of CNS deficiencies associated with metabolic diseases, by retargeting AAV2 to brain endothelia with motifs identified in vivo from phage display libraries.