Gene Knockout And Replacement In Stem Cells

Tech ID: 20792 / UC Case 2008-593-0

Brief Description

It is often advantageous to ascertain the biological purpose of a gene product by "knocking out" that gene and observing the phenotypic consequence(s). This is most often accomplished in whole animal experiments that are costly and take long periods of time related to the gestation period of the animal system. Here we divulge a system where this goal can be accomplished in a short period of time in laboratory cultured animal cells.

Full Description

It is often advantageous to ascertain the biological purpose of a gene product by "knocking out" that gene and observing the phenotypic consequence(s). This is most often accomplished in whole animal experiments that are costly and take long periods of time related to the gestation period of the animal system. Here we divulge a system where this goal can be accomplished in a short period of time in laboratory cultured animal cells. This method is enabled by the fact that every sequence region of a gene optimized using UCI’s proprietary methods (“computationall optimized DNA assembly” or “CODA”) is identified by a unique thermodynamic address. This means that any unique gene X can be CODA-designed and assembled for insertion and stable expression in an animal cell genome in parallel with an siRNA directed against the host cell gene X mRNA but with no sequence homology to the CODA gene X mRNA; thus the expression of the host cell gene X protein product will be inhibited and the expression of the CODA gene X will be promoted. The result is the expression of gene X and its protein product in the place of the cell's own gene X. A novel extension of this replacement method is that it can be performed in pluripotent stem cells from a patient or experimental animal that can subsequently be induced to differentiate into tissue-specific cell types that can be reintroduced into the patient or experimental animal. An example of this application might be the introduction of a normal CODA designed human huntingtin gene into stem cells obtained from a patient afflicted with Huntington's disease wherein the diseased huntingtin gene would be inactivated and the normal CODA human huntingtin gene and its normal protein product would be expressed. These gene replacement cells could then be induced to differentiate into neuronal cells and reintroduced into the Huntington's disease patient. Further gene therapy applications of these methods could involve the replacement of any diseased gene in a patient's stem cells followed by tissue-specific or organ specific implantation to cure a genetic defect such as an inborn error of metabolism or to correct any spontaneous mutation such as ones associated with the onset of cancer.

Suggested uses

An example of this application might be the introduction of a normal CODA designed human huntingtin gene into stem cells obtained from a patient afflicted with Huntington's disease wherein the diseased huntingtin gene would be inactivated and the normal CODA human huntingtin gene and its normal protein product would be expressed. These gene replacement cells could then be induced to differentiate into neuronal cells and reintroduced into the Huntington's disease patient. Further gene therapy applications of these methods could involve the replacement of any diseased gene in a patient's stem cells followed by tissue-specific or organ specific implantation to cure a genetic defect such as an inborn error of metabolism or to correct any spontaneous mutation such as ones associated with the onset of cancer.

Advantages

It is often advantageous to ascertain the biological purpose of a gene product by "knocking out" that gene and observing the phenotypic consequence(s). Here we divulge a system where this goal can be accomplished in a short period of time in laboratory cultured animal cells.

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Keywords

gene knockout

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