Method for inducing DNA synthesis in neurons

Details Method for inducing DNA synthesis in neurons Method for inducing DNA synthesis in neurons

1996-11-18

2000-07-11

Degen, Nancy

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

435455, 435465, 514 44, A61K 4800, C12N 1587, C12N 1588, C12N 1590

Patent

active

060871717

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND AND SUMMARY OF THE INVENTION

The mammalian nervous system has no mechanisms to replace lost neurons with the exception of some regions where neurogenesis continues throughout life (Kaplan et al., Science, 197:1092-1094 (1977); Bayer, Exp. Brain Res., 46: 315-3323 (1982); Altman et al., J. Comp. Neurol., 301:365-381 (1990)). Neurons are born during a narrow window of time and, when differentiated, become blocked at the early G1(G0) phase of the cell cycle (Angevine et al., Nature, 192:766-768 (1961); Takahashi et al., J. Neurocytol., 21:185-197 (1992)). Accordingly, differentiated neurons are not capable of proceeding from the G1(G0) phase to the DNA synthesis phase. Thus, differentiated neurons are not only incapable of synthesizing DNA, but also are incapable of proceeding through the rest of the cell cycle to form new cells (proliferating).
It would be advantageous if one could cause normally differentiated neurons to proceed from the G1(G0) phase to the DNA synthesis phase. By doing so, one could induce proliferation of such neurons to produce new cells.
Moreover, as demonstrated by the present inventors, one can use gene therapy by stably integrating DNA into normally differentiated neurons that have been induced to proceed from the G1(G0) phase to the DNA synthesis phase. Gene therapy involves incorporating nucleic acid into the patient host. In certain applications, the host will express the foreign nucleic acid such that a therapeutic protein is made in the host. Although gene therapy has been used to express nucleic acids in nondifferentiated cells that can proliferate and synthesize nucleic acid, unique issues exist for gene therapy involving transfer of nucleic acid into neurons.
For gene therapy, one should consider factors involved in the delivery of the nucleic acid into the target cell and efficient expression of the nucleic acid in the cell. For therapy involving neurons as the target cells, customarily, one would transfer the nucleic acid in vivo to postmitotic (nonproliferating and fully differentiated) cells. Therapy for diseases of or trauma to the central nervous system may involve use of differentiated neurons as the target cells.
Physical and viral vector methods have been used for gene transfer into neurons of the adult nervous system with limited success. Direct injection of DNA into neurons is possible, however, this is limiting because of the number of cells involved.
Although liposomes, complex ligand DNA conjugates, or viral vectors can deliver DNA to differentiated cells, the subsequent expression of DNA is transient since such cells do not normally synthesize DNA. Gene therapy would be somewhat limited if only transient expression of the transferred DNA was achieved rather than stable functional integration of the transferred DNA because of the shorter periods of expression associated with transient expression.
For many conditions, one desires expression of the DNA over an extended period of time to provide the therapeutic molecule for ongoing treatment. For example, a patient having Parkinson's disease or Alzheimers disease often needs treatment for many years. Functional integration of transferred DNA would provide such long term expression of the therapeutic molecule. Thus, it would be desirable to have stable functional integration of the transferred DNA into neurons for treatment of neurodegenerative diseases and trauma.
The regulation of G1(G0) to DNA synthesis transition involves regulator molecules known as transcription factors. Modification (mainly phosphorylation) of preexisting regulators and transcriptional activation of new genes occurs during this process. Several different protein kinases which form complexes and modify transcription factors are described (Devoto et al., Cell, 68:167-176 (1992), Hunter, Cell, 75:839-841 (1993)). Several transcription factors are described which regulate different steps in G1 to DNA synthesis transition. Retinoblastoma antigen (Rb) (Hamel et al., Molec. Cell. Biol., 12:3431-3438 (1992)), sequence specific transcripti

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