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Gene delivery technology
Overview of brain gene therapy Enzyme replacement in experimental Parkinson's disease with intravenous gene therapy Elimination of ectopic gene expression with brain-specific gene promoters Antisense gene therapy in brain cancer Intravenous RNA interference (RNAi) of the brain OVERVIEW OF BRAIN GENE THERAPY The expectations for gene therapy in the treatment of brain disease is high, because much has been promised with this technology. However, brain gene therapy has not progressed because the gene delivery problem has not been solved. Viral vectors such as adenovirus or herpes virus cause inflammation in the brain. Viral vectors such as retrovirus or adeno-associated virus cause random and permanent integration in the host genome and raise the risk of insertional mutagenesis and cancer. Conventional non-viral vectors such as cationic liposomes or cationic polyplexes aggregate in blood and deposit in the lung without entry into the brain. Neither viral vectors or cationic polyplexes cross the BBB and do not enter brain from blood. The intra-cerebral injection of the gene vector following craniotomy has had limited success, because of the restricted diffusion of the gene from the injection site. What is needed is a non-invasive, non-viral gene delivery technology that enables the widespread expression of an exogenous gene in the brain following a simple intravenous injection. This can be accomplished with a trans-vascular gene delivery technology, which has been developed by scientists at ArmaGen Technologies. GLOBAL EXPRESSION OF EXOGENOUS REPORTER GENES IN THE BRAIN OF RODENTS AND PRIMATES FOLLOWING INTRAVENOUS ADMINISTRATION OF NON-VIRAL FORMULATIONS
BETA-GALACTOSIDASE GENE EXPRESSION IN THE BRAIN The global delivery of an exogenous gene (encoding for the bacterial beta-galacotsidase gene) to the brain is shown by the histochemistry of mouse brain removed 2 days after a single intravenous injection of the gene (see adjacent Figure). This figure shows global expression of an exogenous non-viral gene throughout the entire brain following a single intravenous injection in the mouse. The figure illustrates 'adult transgenics in 24 hours'. ArmaGen Technologies' transformational gene delivery technology has been reduced to practise in mice, rats, and primates. The global expression of the exogenous gene in the brain occurs because the gene enters the brain from blood via the trans-vascular route.Every neuron in the brain is perfused by its own blood vessel. Therefore, the delivery of the gene medicine across the BBB delivers the gene to the 'doorstep' of every neuron in the brain. LUCIFERASE GENE EXPRESSION IN THE BRAIN Non-viral expression plasmids encoding for firefly luciferase have been delivered to the brain of rodents and rhesus monkeys. Gene delivery in the rodent is mediated via the blood-brain barrier (BBB) transferrin receptor (TfR). Gene targeting to the rhesus monkey is mediated via the insulin receptor (IR). Both the TfR and the IR are located on the plasma membrane of both the capillary endothelial cell, which forms the BBB, and the neuron. Therefore, molecular Trojan horses directed at either the TfR or the IR deliver the nanocontainer carrying the gene from blood into the intracellular compartment of the brain cell. The IR also delivers ligands to the nuclear compartment, and the level of gene expression in the primate is 50-fold higher than the level of gene expression in the rat or mouse. The IR delivers ligands directly to the nuclear compartment. Nevertheless, the level of gene expression in rodent brain is sufficient to give a full pharmacological effect, as demonstrated in rodent models of either experimental Parkinsonism or experimental brain cancer. ENZYME REPLACEMENT IN EXPERIMENTAL PARKINSON'S DISEASE WITH INTRAVENOUS GENE THERAPY
ELIMINATION OF ECTOPIC GENE EXPRESSION WITH BRAIN-SPECIFIC GENE PROMOTERS The ectopic expression of the therapeutic gene in organs other than the brain is eliminated when the therapeutic gene is driven by a promoter that is only expressed in the brain. The elimination of ectopic gene expression in non-target organs with organ-specific gene promoters has been demonstrated in mice, rats, and primates with multiple genes. The combination of tissue-specific gene promoters and the gene targeting technology developed by ArmaGen Technologies enables gene developers to achieve expression of therapeutic genes only in target organs following an intravenous administration of the gene. ANTISENSE GENE THERAPY IN BRAIN CANCER Antisense gene therapy of the brain has the potential to cure many brain diseases, including brain cancer and viral infections such as AIDS. In antisense gene therapy, the intent is to produce within the nucleus of the target cell a copy of RNA that is antisense to the target sense mRNA to create the formation of an RNA-RNA duplex, which then blocks expression of the target gene. However, the limiting factor in antisense gene therapy is delivery. It is difficult to achieve effective distribution of the gene vector with a craniotomy-based approach, owing to the limitations of diffusion. Moreover, the use of viral vectors can cause inflammation in the brain leading to demyelination or cause insertional mutagenesis. ArmaGen Technologies has developed a non-invasive, non-viral approach to gene therapy of the brain, whereby the expression plasmid encoding the antisense RNA is encapsulated in a nanocontainer. The therapeutic gene is then targeted across both the BBB and the tumor cell membrane and delivered to the nucleus of the cancer cell within the brain behind the BBB following an intravenous administration. One oncogene target for brain cancer is the epidermal growth factor receptor (EGFR), which is over-expressed in the majority of primary glioblastoma multiforme (GBM), the most malignant form of brain cancer. The EGFR is also oncogenic in up to 70% of all solid cancers outside the brain. Scientists at ArmaGen Technologies have produced expression plasmids that encode for antisense RNA against the human EGFR mRNA. The EGFR antisense gene is delivered to cancer cells behind the BBB in mice with intra-cranial human brain cancer using ArmaGen Technologies' proprietary gene delivery technology. The survival time of mice with experimental, intra-cranial human brain cancer is increased 100% with weekly intravenous antisense gene therapy directed against the human EGFR. The increase in survival is observed even if treatment is delayed until the intracranial brain cancer is large and occupying the entire striatum. These results show that antisense gene therapy works in cancer, providing the gene delivery problem is solved. ArmaGen Technologies' gene delivery technology provides solutions to the delivery problem and enables successful applications of gene therapy in cancer. See Products. INTRAVENOUS RNA INTERFERENCE (RNAi) OF THE BRAIN A new form of antisense therapy is RNA interference (RNAi), which causes gene silencing following the intracellular injection of a short RNA duplex. RNA is unstable in vivo, but the RNA duplex can be synthesized inside the cell following the effective delivery to brain of a non-viral expression plasmid that encodes for the RNA. ArmaGen Technologies' delivery technology enables intravenous RNA interference (RNAi) in brain for the first time. Intravenous RNAi yields a 90% knockdown of brain cancer gene expression, and this knockdown persists for at least 5 days after an intravenous injection of the gene in low doses and small volumes. In addition to brain cancer, RNAi-based gene therapy of the brain can be used to treat viral infections of the brain such as AIDS, as well as inborn errors of metabolism to knock-down pathologic dominant genes. RNAi is an exciting technology, but this technology is simply a new form of gene therapy and, as such, will suffer from the same limitations related to vector delivery that has slowed the general field of gene therapy for years. A gene delivery technology that targets the RNAi expression vector to distant sites in the body behind membrane barriers following an intravenous injection will enable clinical applications of RNAi, and this delivery technology has been developed by ArmaGen Technologies. |
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Many disorders of the brain could be treated, even cured, with gene therapy.
ArmaGen has developed a new form of non-viral
gene transfer to the brain, wherein nanocontainers carrying the plasmid DNA
are targeted to the brain with the same molecular Trojan horses (MTH) that are
used to ferry across the BBB recombinant proteins (see 
The
delivery across the BBB of a therapeutic gene in an experimental model of Parkinsons
disease is shown in the Figure; in this model the level of tyrosine hydroxylase
(TH) in the striatum is completely depleted, as shown by the immunocytochemistry
of the animal treated with TH gene therapy and no molecular Trojan horse (bottom
panel in Figure). However, the level of the TH enzyme in the striatum on the
lesioned side of brain is completely normalized by the intravenous injection
of the TH gene (top panel in Figure). The restoration of the striatal TH enzyme
is associated with a normalization of the motor abormalities associated with
experimental Parkinsons disease, such as apomorphine-induced rotation behavior.
The expression of the TH gene in the brain is confined to the striatum, and
is not expressed in the cortex. Neuronal expression of the exogenous gene is
verified by confocal microscopy. Therefore, TH gene therapy with this approach
may have minimal side effects, unlike other forms of dopamine replacement therapy
of Parkinsons disease, where dopapmine production in regions of the brain other
than the striatum is also increased.Dose response studies show that the striatal
TH gene expression is normalized with the delivery of only 5-10 plasmid molecules
per brain cell, which illustrates the very high efficiency of neuronal transfection
with this gene delivery technology. The therapeutic effect in rats lasts a week,
and is expected to persist for several weeks in the primate, where the level
of gene expression is 50-fold higher than in the rodent. Monthly intravenous
TH gene therapy can maintain TH levels in the normal range in patients with
Parkinson's disease. The neurodegeneration of PD can be slowed by the combined
gene therapy of TH and a neurotrophin that protects neurons in the nigro-striatal
tract.