Biomedical subjects

Xandra O Breakefield

Publications and source records attributed to Xandra O Breakefield.

At least 19 recordsLinked to original sources

PubMed2025

AstroGreen transgenic mouse illuminates the trafficking of astrocyte-derived extracellular vesicles.

Astrocytes interact with neighboring cells by releasing extracellular vesicles (EVs). Tools to study astrocyte EV-mediated communication with other brain cells in vivo are essential. In this study, we crossed the Exomap1 transgenic mouse expressing Cre-activated human-specific CD81 (HsCD81) fused to the fluorescent protein mNeonGreen (HsCD81mNG), to a transgenic mouse expressing Cre under the astrocyte-expressing GFAP promoter resulting in Exomap1::Gfap-Cre mice, referred to here as AstroGreen. We characterized HsCD81mNG-expressing astrocytes and shedded EVs loaded with HsCD81mNG and Cre, both in vitro and in mouse brains. Using this model, we show that HsCD81mNG can be used to track EV content, production, and functional Cre transfer in vitro and in the brain, allowing evaluation of the interaction of astrocytes with neighboring cells mediated by EVs. We anticipate that this model will improve our understanding of astrocytes transferring EVs within their surroundings during normal physiological processes and in the context of neuropathological conditions.

Animals↗

PubMed2014

Gene therapy for the nervous system: challenges and new strategies.

Current clinical treatments for central nervous system (CNS) diseases, such as Parkinson's disease and glioblastoma do not halt disease progression and have significant treatment morbidities. Gene therapy has the potential to "permanently" correct disease by bringing in a normal gene to correct a mutant gene deficiency, knocking down mRNA of mutant alleles, and inducing cell-death in cancer cells using transgenes encoding apoptosis-inducing proteins. Promising results in clinical trials of eye disease (Leber's congenital aumorosis) and Parkinson's disease have shown that gene-based neurotherapeutics have great potential. The recent development of genome editing technology, such as zinc finger nucleases, TALENS, and CRISPR, has made the ultimate goal of gene correction a step closer. This review summarizes the challenges faced by gene-based neurotherapeutics and the current and recent strategies designed to overcome these barriers. We have chosen the following challenges to focus on in this review: (1) delivery vehicles (both virus and nonviral), (2) use of promoters for vector-mediated gene expression in CNS, and (3) delivery across the blood-brain barrier. The final section (4) focuses on promising pre-clinical/clinical studies of neurotherapeutics.

Animals↗

PubMed2006

Effects of genetic variations in the dystonia protein torsinA: identification of polymorphism at residue 216 as protein modifier.

Four naturally occurring sequence variations have been found in the coding region of the DYT1 gene encoding torsinA. One of these, a 3 bp (DeltaGAG) deletion, underlies dominantly inherited cases of early-onset torsion dystonia. Others, including a single nucleotide polymorphism that replaces aspartic acid (D) at residue 216 with histidine (H) in 12% of normal alleles and two other rare deletions, have not been clearly associated with disease. To gain insight into how these sequence variations affect torsinA, we used the structure of the related protein ClpB to provide a model of torsinA's AAA+ domain. Motifs important for ATP hydrolysis-sensor 1 and sensor 2-were identified, mutagenized and used to validate predictions of this model. Inspection revealed that the DeltaGAG deletion associated with dystonia removes one residue from an alpha-helix in the C-terminal portion of the AAA+ domain. The resulting distortion in torsinA structure may underlie this mutant's known tendency to produce ER-derived inclusions as well as its proposed loss of function. The D/H polymorphism at residue 216 falls in the N-terminal portion of the AAA+ domain near the sensor 1 motif. Surprisingly, cells expressing torsinA with the polymorphic histidine developed inclusions similar to those associated with DeltaGAG-torsinA, indicating that this change may also affect torsinA structure. Introducing H216 into DeltaGAG-torsinA reduced its tendency to form inclusions, suggesting that the two changes offset each other. Our findings point to a structural basis for the defects associated with the disease-linked DeltaGAG deletion in torsinA. They also suggest possible connections between the allelic polymorphism at residue 216 and the penetrance of DYT1 dystonia, as well as a possible role for this polymorphism in related disease states.

Adenosine Triphosphatases↗

PubMed2006

Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector.

Oncolytic viral therapy provides a promising approach to treat certain human malignancies. These vectors improve on replication-deficient vectors by increasing the viral load within tumors through preferential viral replication within tumor cells. However, the inability to efficiently propagate throughout the entire tumor and infect cells distant from the injection site has limited the capacity of oncolytic viruses to achieve consistent therapeutic responses. Here we show that the spread of the oncolytic herpes simplex virus (HSV) vector MGH2 within the human melanoma Mu89 is limited by the fibrillar collagen in the extracellular matrix. This limitation seems to be size specific as nanoparticles of equivalent size to the virus distribute within tumors to the same extent whereas smaller particles distribute more widely. Due to limited viral penetration, tumor cells in inaccessible regions continue to grow, remaining out of the range of viral infection, and tumor eradication cannot be achieved. Matrix modification with bacterial collagenase coinjection results in a significant improvement in the initial range of viral distribution within the tumor. This results in an extended range of infected tumor cells and improved virus propagation, ultimately leading to enhanced therapeutic outcome. Thus, fibrillar collagen can be a formidable barrier to viral distribution and matrix-modifying treatments can significantly enhance the therapeutic response.

Animals↗

PubMed2006

Developmental patterns of torsinA and torsinB expression.

Early onset torsion dystonia is characterized by involuntary movements and distorted postures and is usually caused by a 3-bp (GAG) deletion in the DYT1 (TOR1A) gene. DYT1 codes for torsinA, a member of the AAA+ family of proteins, implicated in membrane recycling and chaperone functions. A close relative, torsinB may be involved in similar cellular functions. We investigated torsinA and torsinB message and protein levels in the developing mouse brain. TorsinA expression was highest during prenatal and early postnatal development (until postnatal day 14; P14), whereas torsinB expression was highest during late postnatal periods (from P14 onwards) and in the adult. In addition, significant regional variation in the expression of the two torsins was seen within the developing brain. Thus, torsinA expression was highest in the cerebral cortex from embryonic day 15 (E15)-E17 and in the striatum from E17-P7, while torsinB was highest in the cerebral cortex between P7-P14 and in the striatum from P7-P30. TorsinA was also highly expressed in the thalamus from P0-P7 and in the cerebellum from P7-P14. Although functional significance of the patterns of torsinA and B expression in the developing brain remains to be established, our findings provide a basis for investigating the role of torsins in specific processes such as neurogenesis, neuronal migration, axon/dendrite development, and synaptogenesis.

Age Factors↗

PubMed2006

Metabolic biotinylation of cell surface receptors for in vivo imaging.

We have developed a versatile, potent technique for imaging cells in culture and in vivo by expressing a metabolically biotinylated cell-surface receptor and visualizing it with labeled streptavidin moieties. The recombinant reporter protein, which incorporates a biotin acceptor peptide (BAP) between an N-terminal signal sequence and a transmembrane domain, (BAP-TM) was efficiently biotinylated by endogenous biotin ligase in mammalian cells with the biotin displayed on the cell surface. Tumors expressing the BAP-TM have high sensitivity for magnetic resonance and fluorescence tomographic imaging in vivo after intravascular injection of streptavidin conjugated to magnetic nanoparticles or fluorochromes, respectively. Moreover, streptavidin-horseradish peroxidase conjugates in conjunction with a peroxidase-sensitive gadolinium agent further increased and prolonged the magnetic resonance signal. This BAP-TM allows noninvasive real-time imaging of any cell type transduced to express this reporter protein in culture or in vivo.

Base Sequence↗

PubMed2006

Treatment of schwannomas with an oncolytic recombinant herpes simplex virus in murine models of neurofibromatosis type 2.

Gene therapy for schwannomas was evaluated in two mouse models of neurofibromatosis type 2 (NF2): (1) a transgenic model in which mice express a dominant mutant form of merlin and spontaneously develop schwannomas, and (2) a xenograft model in which human schwannoma tissue is implanted subcutaneously into immune- compromised mice. In both models, schwannoma volumes were monitored by magnetic resonance imaging (MRI) and showed strong gadolinium enhancement typical of these tumors in humans. Both types of tumor were positive for the Schwann cell marker S100, and highly infectable with herpes simplex virus (HSV) vectors. Schwannomas were injected with an oncolytic HSV-1 recombinant virus vector, G47Delta, which has deletions in genes for ribonucleotide reductase (ICP6), gamma34.5, and ICP47. In the NF2 transgenic model, schwannomas were reduced by more than half their original size by 10 days after infection. In the case of subcutaneous schwannoma xenografts, reduction in size after infection occurred more slowly, with a mean reduction of onethird by 42 days after treatment. Schwannomas injected with control vehicles continued to grow slowly over time in both schwannoma models. These studies demonstrate the ability of an oncolytic recombinant HSV vector to reduce the volume of schwannoma tumors in NF2 tumor models in mice and extend the possible therapeutic applications of oncolytic vectors for benign tumors to reduce mass while minimizing nerve damage.

Animals↗

PubMed2006

HSV amplicon vectors for cancer therapy.

HSV amplicon vectors provide a unique tool in the armamentarium of weapons for treatment of cancer. Their large capacity (up to 150 kb) allows incorporation of multiple and large transgenes, including whole gene loci, as well as components of other viruses to control the fate of transgenes in the host cells. Means have been developed to achieve heritable transmission of transgenes in tumor cells by episomal replication or genomic integration. Therapeutic transgenes incorporated into amplicon vectors have included anti-angiogenic agents, immune enhancing proteins, prodrug activating enzymes, and apoptosis-inducing factors, as well as inhibitory RNAs for tumor-associated messages. Perks of this vector system include the ability to combine amplicon vectors with oncolytic HSV recombinant vectors to extend the therapeutic range and to target non-dividing as well as dividing tumor cells. Tumor vaccination is favored by the high infectivity of dendritic antigen-presenting cells with HSV vectors, and the vectors themselves appear to have intrinsic immune enhancing properties. Promoter manipulation can be used to target therapeutic gene expression to specific tumor cell types and to achieve drug regulated transgene expression. Further, amplicon vectors can be used to convert tumor cells into packaging cells for retrovirus and adeno-associated virus vectors, thus generating vectors on site. Amplicon vectors have also proven to be a versatile tool to explore imaging modalities to monitor gene delivery and tumor responses to therapeutic intervention.

Animals↗

PubMed2005

Dystonia-causing mutant torsinA inhibits cell adhesion and neurite extension through interference with cytoskeletal dynamics.

Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms-recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA-the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.

Actins↗

PubMed2005

RNAi blocks DYT1 mutant torsinA inclusions in neurons.

Early onset generalized dystonia is a dominantly inherited movement disorder caused by neuronal dysfunction without an apparent loss of neurons. The same single mutation (GAG deletion) causes most cases and results in loss of a glutamic acid (E) in the carboxy terminal region of torsinA (Delta302/303). To model the neuronal involvement, adult rat primary sensory dorsal root ganglion neurons in culture were infected with lentivirus vectors expressing human wild-type or mutant torsinA. Expression of the mutant protein resulted in formation of torsinA-positive perinuclear inclusions. When the cells were co-infected with lentivirus vectors expressing the mutant torsinA message and a shRNA selectively targeting this message, inclusion formation was blocked. Vector-delivered siRNAs have the potential to decrease the adverse effects of this mutant protein in neurons without affecting wild-type protein.

Animals↗

PubMed2005

Impaired motor learning in mice expressing torsinA with the DYT1 dystonia mutation.

Primary early-onset generalized dystonia is an autosomal dominant disorder caused by a deletion (DeltaGAG) in the DYT1 gene encoding torsinA. The gene defect has incomplete penetrance, with approximately 30% of carriers developing clinically evident dystonia. We describe lines of transgenic mice that express either human mutant torsinA (hMT) or human wild-type (hWT) torsinA. All mice demonstrated moderately increased levels of torsinA in the brain by Western blot analysis and normal subcellular distribution of torsinA in neurons by confocal microscopy. No animals had dystonic features. However, mice overexpressing hMT, but not hWT, torsinA displayed a reduced ability to learn motor skills in an accelerating rotarod paradigm. This pattern resembles the impaired motor sequence learning demonstrated in human nonmanifesting carriers of the DeltaGAG mutation. Open-field testing showed no differences in spontaneous activity between transgenic mice and their nontransgenic littermates, indicating that mice overexpressing hMT torsinA did not develop overtly abnormal motor behavior. Together, these data suggest that these transgenic mice provide a useful model of the DeltaGAG carrier state that can be used to probe genetic and environmental factors that can trigger the dystonic state.

Animals↗

PubMed2005

Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression.

Despite many refinements in current therapeutic strategies, the overall prognosis for a patient with glioblastoma is dismal. Neural precursor cells (NPCs) are capable of tracking glioma tumors and thus could be used to deliver therapeutic molecules. We have engineered mouse NPCs to deliver a secreted form of tumor necrosis factor-related apoptosis-inducing ligand (S-TRAIL); S-TRAIL is optimized to selectively kill neoplastic cells. Furthermore, we have developed means to simultaneously monitor both the migration of NSCs toward gliomas and the changes in glioma burden in real time. Using a highly malignant human glioma model expressing Renilla luciferase (Rluc), intracranially implanted NPC-FL-sTRAIL expressing both firefly luciferase (Fluc) and S-TRAIL was shown to migrate into the tumors and have profound antitumor effects. These studies demonstrate the potential of NPCs as therapeutically effective delivery vehicles for the treatment of gliomas and also provide important tools to evaluate the migration of NPCs and changes in glioma burden in vivo.

Animals↗

PubMed2005

Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo.

Photoproteins have played a major role in advancing our understanding of biological processes. A broader array of biocompatible, nontoxic, and novel reporters can serve to expand this potential. Here we describe the properties of a luciferase from the copepod marine organism Gaussia princeps. It is a monomeric protein composed of 185 aa (19.9 kDa) with a short coding sequence (555 bp) making it suitable for viral vectors. The humanized form of Gaussia luciferase (hGLuc) was efficiently expressed in mammalian cells following delivery by HSV-1 amplicon vectors. It was found to be nontoxic and naturally secreted, with flash bioluminescence characteristics similar to those of other coelenterazine luciferases. hGLuc generated over 1000-fold higher bioluminescent signal intensity from live cells together with their immediate environment and over 100-fold higher intensity from viable cells alone (not including secreted luciferase) or cell lysates, compared to humanized forms of firefly (hFLuc) and Renilla (hRLuc) luciferases expressed under similar conditions. Furthermore, hGLuc showed 200-fold higher signal intensity than hRLuc and intensity comparable to that of hFLuc in vivo under standard imaging conditions. Gaussia luciferase provides a sensitive means of imaging gene delivery and other events in living cells in culture and in vivo, with a unique combination of features including high signal intensity, secretion, and ATP independence, thus being able to report from the cells and their environment in real time.

Animals↗

PubMed2005

In vivo imaging of S-TRAIL-mediated tumor regression and apoptosis.

Therapeutic proteins with specific effector functions play an increasingly important role in drug therapy. For example, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) predominantly kills cancer cells, while sparing normal cells. Here, we report the use of a secreted version of TRAIL as a therapeutic protein that induces apoptosis and kills surrounding cells in vivo, thus resulting in the dramatic reduction of glioma burden in mouse tumor models. Using a caspase-3-activatable aminoluciferin, we were able to show the induction of apoptosis specifically in S-TRAIL vector-infected gliomas. We also show that S-TRAIL-mediated apoptosis and resulting changes in tumor burden can be imaged in the same animal by dual-substrate bioluminescence imaging. The use of S-TRAIL as a therapeutic protein and the ability to image noninvasively both apoptosis and any other cellular events in real time have important clinical implications.

Animals↗

PubMed2004

TorsinA in the nuclear envelope.

Early-onset torsion dystonia, a CNS-based movement disorder, is usually associated with a single amino acid deletion (Delta E302/303) in the protein torsinA. TorsinA is an AAA+ ATPase in the endoplasmic reticulum, but what it does is unknown. Here, we use torsinA mutants with defects in ATP hydrolysis (E171Q, ATP-bound) and ATP binding (K108A, ATP-free) to probe torsinA's normal cellular function. Surprisingly, ATP-bound torsinA is recruited to the nuclear envelope (NE) of transfected cells, where it alters connections between inner and outer nuclear membranes. In contrast, ATP-free torsinA is diffusely distributed throughout the endoplasmic reticulum and has no effect on the NE. Among AAA+ ATPases, affinity for substrates is high in the ATP-bound and low in the ATP-free state, leading us to propose that component(s) of the NE may be substrates for torsinA. We also find that the disease-promoting Delta E302/303 mutant is in the NE, and that this relocalization, as well as the mutant's previously described ability to induce membranous inclusions, is eliminated by the K108A ATP-binding mutation. These results suggest that changes in interactions involving torsinA in the NE could be important for the pathogenesis of dystonia and point to torsinA and related proteins as a class of ATPases that may operate in the NE.

Adenosine Triphosphatases↗

PubMed2004

Inducible release of TRAIL fusion proteins from a proapoptotic form for tumor therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill neoplastic cells and control of its activity could enhance tumor therapy. We have developed means to control the secretion of a novel recombinant (r) TRAIL fusion protein using a viral protease. This system uses the endoplasmic reticulum (ER) as a storage depot for rTRAIL, because TRAIL acts by binding to its cognate receptors on the cell surface. We have engineered two TRAIL variants: (a) a secretable form that enhances apoptosis via a bystander effect; and (b) an ER-targeted TRAIL that is retained in the ER until selectively released by the viral protease. Gene delivery can be monitored in vivo by systemic administration of a near infrared fluorescent (NIRF) probe activated by the protease. This study serves as a template for design of recombinant proteins to enhance and control apoptosis of tumor cells via specific viral proteases and for use of viral proteases as in vivo reporters for cancer therapy.

Animals↗

PubMed2004

Identification of a novel gene (HSN2) causing hereditary sensory and autonomic neuropathy type II through the Study of Canadian Genetic Isolates.

Hereditary sensory and autonomic neuropathy (HSAN) type II is an autosomal recessive disorder characterized by impairment of pain, temperature, and touch sensation owing to reduction or absence of peripheral sensory neurons. We identified two large pedigrees segregating the disorder in an isolated population living in Newfoundland and performed a 5-cM genome scan. Linkage analysis identified a locus mapping to 12p13.33 with a maximum LOD score of 8.4. Haplotype sharing defined a candidate interval of 1.06 Mb containing all or part of seven annotated genes, sequencing of which failed to detect causative mutations. Comparative genomics revealed a conserved ORF corresponding to a novel gene in which we found three different truncating mutations among five families including patients from rural Quebec and Nova Scotia. This gene, termed "HSN2," consists of a single exon located within intron 8 of the PRKWNK1 gene and is transcribed from the same strand. The HSN2 protein may play a role in the development and/or maintenance of peripheral sensory neurons or their supporting Schwann cells.

Amino Acid Sequence↗

PubMed2004

Bone marrow transplantation restores immune system function and prevents lymphoma in Atm-deficient mice.

Ataxia-telangiectasia (A-T) is a human autosomal recessive disease caused by mutations in the gene encoding ataxia-telangiectasia mutated (ATM). A-T is characterized by progressive cerebellar degeneration, variable immunodeficiency, and a high incidence of leukemia and lymphoma. Recurrent sino-pulmonary infections secondary to immunodeficiency and hematopoietic malignancies are major causes of morbidity and mortality in A-T patients. In mice, an introduced mutation in Atm leads to a phenotype that recapitulates many of the symptoms of A-T, including immune system abnormalities and susceptibility to malignancy. Here we show that the replacement of the bone marrow compartment in Atm knockout mice (Atm(-/-)) using a clinically relevant, nonmyeloablative host-conditioning regimen can be used to overcome the immune deficiencies and prevent the malignancies observed in these mice. Therefore, bone marrow transplantation may prove to be of therapeutic benefit in A-T patients.

Animals↗