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1.
Sci Rep ; 11(1): 1934, 2021 01 21.
Article in English | MEDLINE | ID: mdl-33479314

ABSTRACT

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood-brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.


Subject(s)
Brain/drug effects , Brain/metabolism , Dependovirus/genetics , Liver/drug effects , Animals , Blood-Brain Barrier/drug effects , Brain/diagnostic imaging , Genetic Therapy , Genetic Vectors/therapeutic use , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/pharmacology , Humans , Injections, Intravenous , Liver/diagnostic imaging , Magnetic Resonance Imaging , Mice , Neurons/drug effects , Promoter Regions, Genetic , Synapsins/chemistry , Synapsins/pharmacology , Tissue Distribution , Transduction, Genetic , Ultrasonography
2.
Theranostics ; 9(26): 8127-8137, 2019.
Article in English | MEDLINE | ID: mdl-31754385

ABSTRACT

Gene therapy can be designed to efficiently counter pathological features characteristic of neurodegenerative disorders. Here, we took advantage of the glial fibrillary acidic protein (GFAP) promoter to preferentially enhance transgene expression near plaques composed of amyloid-beta peptides (Aß), a hallmark of Alzheimer's disease (AD), in the TgCRND8 mouse model of amyloidosis. Methods: The delivery of intravenously injected recombinant adeno-associated virus mosaic serotype 1/2 (rAAV1/2) to the cortex and hippocampus of TgCRND8 mice was facilitated using transcranial MRI-guided focused ultrasound in combination with microbubbles (MRIgFUS), which transiently and locally increases the permeability of the blood-brain barrier (BBB). rAAV1/2 expression of the reporter green fluorescent protein (GFP) under a GFAP promoter was compared to GFP expression driven by the constitutive human beta actin (HBA) promoter. Results: MRIgFUS targeting the cortex and hippocampus facilitated the entry of rAAV1/2 and GFP expression under the GFAP promoter was localized to GFAP-positive astrocytes. Adjacent to Aß plaques where GFAP is upregulated, the volume, surface area, and fluorescence intensity of the transgene GFP were greater in rAAV1/2-GFAP-GFP compared to rAAV1/2-HBA-GFP treated animals. In peripheral organs, GFP expression was particularly strong in the liver, irrespective of the promoter. Conclusion: The GFAP promoter enhanced transgene expression in proximity of Aß plaques in the brain of TgCRND8 mice, and it also resulted in significant expression in the liver. Future gene therapies for neurological disorders could benefit from using a GFAP promoter to regulate transgene expression in response to disease-induced astrocytic reactivity.


Subject(s)
Gene Transfer Techniques , Glial Fibrillary Acidic Protein , Plaque, Amyloid/pathology , Promoter Regions, Genetic , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Animals , Astrocytes/metabolism , Disease Models, Animal , Gene Expression , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Liver/metabolism , Mice , Mice, Transgenic , Plaque, Amyloid/metabolism , Transgenes
3.
Methods Mol Biol ; 1950: 177-197, 2019.
Article in English | MEDLINE | ID: mdl-30783974

ABSTRACT

Recombinant adeno-associated viral (rAAV) vectors are a promising tool for therapeutic gene delivery to the brain. However, the delivery of rAAVs across the blood-brain barrier (BBB) and entry into the brain remains a major challenge for rAAV-based gene therapy. To circumvent this limitation, transcranial MRI-guided focused ultrasound (MRIgFUS) combined with intravenously injected microbubbles has been used to transiently and reversibly increase BBB permeability in targeted brain regions. Systemic administration of rAAVs at the time of sonication with focused ultrasound (FUS) facilitates the passage of rAAVs through the BBB and into the brain parenchyma. We and others have demonstrated that FUS-mediated rAAV delivery to the brain results in efficient transduction and transgene expression in vivo. Using this approach, the dose of intravenously injected rAAV variants that can cross the BBB can be reduced by 100 times, achieving significant transgene expression in the brain parenchyma with reduced peripheral transduction. Moreover, this strategy can be used to deliver rAAV variants that do not cross the BBB from the blood to selected brain regions. Here, we provide a detailed protocol for FUS-induced BBB permeability for targeted rAAV delivery to the brain of adult mice and rats.


Subject(s)
Brain/diagnostic imaging , Brain/metabolism , Dependovirus/genetics , Gene Transfer Techniques , Genetic Vectors/genetics , Magnetic Resonance Imaging , Neuronavigation , Ultrasonography , Animals , Biological Transport , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/radiation effects , Gene Expression , Genes, Reporter , Genetic Vectors/administration & dosage , Magnetic Resonance Imaging/methods , Mice , Neuronavigation/methods , Permeability/radiation effects , Rats , Transgenes , Ultrasonography/methods
4.
Discoveries (Craiova) ; 5(3): e78, 2017 Sep 30.
Article in English | MEDLINE | ID: mdl-32309596

ABSTRACT

The presence of protein aggregates in the brain is a hallmark of neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). Considerable evidence has revealed that the pathological protein aggregates in many neurodegenerative diseases are able to self-propagate, which may enable pathology to spread from cell-to-cell within the brain. This property is reminiscent of what occurs in prion diseases such as Creutzfeldt-Jakob disease. A widely recognized feature of prion disorders is the existence of distinct strains of prions, which are thought to represent unique protein aggregate structures. A number of recent studies have pointed to the existence of strains of protein aggregates in other, more common neurodegenerative illnesses such as AD, PD, and related disorders. In this review, we outline the pathobiology of prion strains and discuss how the concept of protein aggregate strains may help to explain the heterogeneity inherent to many human neurodegenerative disorders.

6.
J Mol Model ; 18(9): 4131-9, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22527278

ABSTRACT

Enterohemorrhagic (EHEC) and enteroaggregative (EAEC) are two pathotypes of diarrheagenic Escherichia coli. EAEC strains express adhesins called aggregate adherence fimbriae (AAFs) which the bacteria use to adhere to intestinal mucosa. EHEC virulence factor is Shiga toxin which belongs to the AB5 toxin family. B subunit, the nontoxic part of Shiga toxin (StxB), forms a homo pentamer and is responsible for binding to target cells. StxB has recently been proven to have adjuvant activity. In the current study we fused StxB encoding gene to 3' end of genes encoding two variants of AAFs, i.e., AAF/I and AAF/II. The in silico studies on tertiary structure and biochemical characteristics of Shiga toxin A subunit (StxA) revealed more resemblance to AAF/II than AAF/I. The constructs were prepared in a way that StxB could imitate its natural structure (pentamer formation) and its position (C-terminus) in the native toxin complex. The expression of these constructs showed the formation of AAF/II-B as a protein complex but with lower molecular mass than its expected size. In contrast, the AAF/I-B complex was not formed. Overall, the results of in silico studies and expression experiments together revealed that despite AAF/II-B expression, StxB failed to form pentamer. Therefore the observed protein complex has lower molecular mass. Since StxB is bound to AAF/II through disulfide bond, this bond prevents pentamer formation of StxB. However, due to the lack of disulfide bond between AAF/I and StxB, no protein complex is formed, thus StxB maintains its pentamer structure.


Subject(s)
Adhesins, Escherichia coli/metabolism , Computational Biology/methods , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Fimbriae Proteins/metabolism , Models, Molecular , Peptides/metabolism , Shiga Toxins/metabolism , Adhesins, Escherichia coli/chemistry , Bacterial Adhesion , Blotting, Western , Disulfides/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli Proteins/chemistry , Fimbriae Proteins/chemistry , Peptides/chemistry , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Shiga Toxins/chemistry
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