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The Breakefield laboratory has expertise in molecular genetics and neuroscience focusing on understanding and treating diseases of the nervous system.
Their recent work includes characterization of the nucleic acid content of exosomes (microvesicles) released by tumor cells, including their use as biomarkers in the serum of cancer patients for genetic evaluation of tumors, their ability to carry out horizontal gene transfer to other cells to promote tumor growth; and their potential for tumor therapy.
In addition, since identifying the gene for early onset torsion dystonia in 1997, they have worked on understanding the function of the defective protein, torsinA.
They have found that torsinA, an AAA+ ATPase in the lumen of the nuclear envelope and endoplasmic reticulum, is involved in positioning of the nucleus during cell migration and in chaperoning proteins in the secretory pathway.
They are also actively involved in use of viral vectors, including lentivirus and AAV and for gene therapy in the nervous system.
Xandra O. Breakefield, PhD
Leonora Balaj, Post Doctorate Fellow
My research focuses on the utilization and development of adeno-associated virus (AAV) vectors for efficient gene transfer to target organs of the body, in particular the central nervous system (CNS).
AAV is currently the lead candidate for gene therapy to the brain, as it efficiently delivers transgenes to neurons after direct injection. Some AAV vectors can also deliver genes to the brain after vascular injection. This is a preferred route of injection as it is non-invasive and has the potential to provide widespread access of virus vector to the brain.
Some limitations which may hamper the general use of AAV for human gene therapy via the vascular route are neutralizing antibodies to the virus itself, uptake by non-target organs such as the liver and low efficiency of crossing the blood-brain barrier.
Our goal is to overcome these barriers using different strategies including: (1) our recent discovery that AAV vectors can associate with endogenous lipid structures called microvesicles and (2) genetic engineering of the virus.
Our research has centered on understanding and treating diseases of the nervous system. My particular expertise is in molecular genetics, including human genetics, gene therapy and biomarker development with a focus on movement disorders and brain tumors. Since identifying the gene for early onset torsion dystonia in 1997, we have worked on understanding the function of the defective protein, torsinA. We have shown that torsinA, an AAA+ ATPase in the lumen of the nuclear envelope and endoplasmic reticulum of all cells is involved in positioning of the nucleus during cell migration and in chaperoning proteins in the secretory pathway. We are currently focusing on identifying the gene responsible for X-linked dystonia-parkinsonism and its relationship to other forms of dystonia. We have also used viral vectors, including retrovirus, HSV and AAV, as well as migratory neuroprecursor cells, for delivery of genes encoding therapeutic proteins and pro-drug activating enzymes for experimental tumors of the nervous system. In addition, we are characterizing the nucleic acid content of extracellular vesicles released by tumor cells, including their use as biomarkers in the serum, their ability to carry out horizontal cell communication; and their potential for therapy of neurologic diseases.
A major focus of the Breakefield laboratory is on design and development of HSV amplicon vectors for stable, non-toxic delivery of genes in vivo. These vectors have a transgene capacity of 150 kb allowing them to carry entire genes, including regulatory elements and splice junctions. They express no viral genes and elicit only a mild immune response. They have proven to be highly effective in gene delivery in the nervous system in models of lysosomal storage diseases, ataxia telangiectasia, neurofibromatosis and brain tumors.
Vectors for gene delivery are derived from herpes simplex virus type 1 (the common cold sore virus). These virions provide an efficient means to shuttle genes to the cell nucleus. The virions bind to and enter most cells through fusion of the virion envelope with the plasma membrane. Viron capsids and associated tegument proteins are then taken to the cell nucleus via microtubule mediated transport. At the nucleopores the capsid opens and viral DNA is threaded into the nucleus.
The virion has four compartments, all of which can be used as delivery vehicles. The envelope can be modified so that it targets infection to specific cell types (Grandi et al., 2004). Proteins in the tegument, such as VP16, can be fused with reporter proteins, such as GFP (Bearer et al., 2000) or functional proteins to temporarily alter the physiology of infected cells. Capsid proteins can also be modified as fusion proteins and up to 150 kb of foreign DNA can be incorporated in the capsids for delivery.
In infected cells the viral DNA is replicated from origins of replication, ori, as a rolling circle with 150 kb lengths being placed in the capsids in a capsid-full state with cleavage at pac signals. Plasmid DNA bearing ori and pac signals are packaged as concatenates in virions in the presence of viral functions and can be prepared as helper virus free stocks (Saeki et al., 2003).
Several modifications have been incorporated into amplicon plasmids to control the fate of the DNA when it enters the cell nucleus. These include incorporation of the latent origin of DNA replication and EBNA1 sequences from Epstein Barr virus that allow replication of amplicon DNA as an extrachromosomal element (Sena-Esteves et al., 2002; Hampl et al., 2003). Further, elements of retrovirus and AAV have been incorporated into amplicon vectors such that infected cells are able to generate retrovirus vectors and integrate genes into the host cell genome at specific sites (Oehmig et al., 2004).
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