Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-07-06
2003-02-18
Yucel, Remy (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C436S071000, C435S320100, C530S350000
Reexamination Certificate
active
06521456
ABSTRACT:
The present invention relates to a nuclear transport agent, to a gene transfer system comprising said nuclear transport agent, to a method for transporting DNA into the nucleus of eukaryotic cells using said nuclear transport agent and to the use of said nuclear transport agent in gene therapy for treating cancer, viral infections, diseases of the nervous system, graft rejection and monogenic or polygenic hereditary diseases.
The active transport into the nucleus is necessary for the transfer of genetic material into all cells that do not divide in the period before the intended expression of the genetic material. A nuclear transport system for nucleic acids is very important because it facilitates the efficient transfer of DNA into those cells that divide rarely or not at all (Dowty et al., 1995, Wilke et al., 1996). Most primary cells belong to this group. Primary cells are of highest scientific interest for two reasons. Firstly, said cells that have freshly been isolated from an organism reflect the functional state of the cell type much better than cell lines derived therefrom. Secondly, they are the target cells for gene therapy. In addition, a nuclear transport system increases the efficiency of DNA transfer into established cell lines by enabling also those cells to express transferred genetic material, which have not divided in the period of time between start of transfer and analysis.
Genetic material is active in the nucleus. The transport therein can either occur coincidentally during cell division when the nuclear envelope temporarily disintegrates in the course of mitosis or it has to take place actively.
1) Nuclear Proteins are Transported into the Nucleus by Means of Nuclear Localization Signals
The double membrane that envelops the nucleus has pores. Little molecules can pass through these pores by diffusion. In order to be able to enter the nucleus, proteins larger than about 50 kDa need a nuclear localization signal (NLS) that has to be recognized by the transport machinery. Typically, a sufficient signal consists of four to eight amino acids, is rich in the positive amino acids arginine and lysine and contains prolines. It is strongly conserved in evolution so that mammalian NLS are also functional in yeast. Heterologous NLS can also be used as a tool to transport target molecules into the nucleus. For this purpose, NLS can be incorporated into the sequences of cytoplasmic proteins at relatively random positions or can be coupled chemically to proteins or even gold particles (reviewed in Görlich, 1998).
2) Many Viruses Use Nuclear Protein Transport Machinery of the Cell for the Transport of Their DNA into the Nucleus
HIV and other lentiviruses that are able to infect resting cells use viral proteins and the cellular transport machinery to transfer their DNA into the nucleus. The NLS in Vpr and matrix protein of the HIV pre-integration complex (Gallay et al., 1996) are essential for the infection of cells that do not divide (Naldini et al., 1996). Although little is known about how viruses transfer their genomes into the nucleus, the help of viral structural proteins containing NLS might even be a general principle. This is also suggested by the following observations: A specific mutation in the HSV capsid protein prevents the transport of viral DNA into the nucleus (Batterson et al., 1983). Adenovirus DNA is transported into the nucleus together with the hexon protein of the disintegrated capsid (Greber et al., 1993). The transport of SV40 DNA into the nucleus is mediated by a viral protein (probably Vp3) that remains associated with DNA (Nakanishi et al., 1996). Two bacterial proteins containing NLS are responsible for the import of
Agrobacterium tumefaciens
T-DNA into plant nuclei (Citovsky et al., 1994).
Due to the ability of some viruses to infect resting cells, mutant variants of, for example, HIV, adenovirus and herpes virus are used as DNA transfer vehicles for the development of gene therapy approaches. Firstly, this involves the risk of immunological reactions to virus components (Friedmann, 1994, 1996) and, secondly, helper cell lines are used in such systems for which the release of less mutated virus genomes cannot be excluded. Moreover, the handling of these systems is difficult.
Several artificial systems have been described that are supposed to increase transfection efficiency by means of peptides or proteins containing nuclear localization signals.
A) Proteins
Kaneda et al. (1989) and Dzau and Kaneda (1997, U.S. Pat. No. 5,631,237) describe a gene transfer system that is based on the use of Sendai virus, liposomes and added proteins that are meant to support nuclear transport of DNA. For this purpose, the group used HMG-1 (high mobility group 1 protein), a basic non histone protein of chromatin which binds to DNA. HMG-1 binds to DNA through a long basic region. It is localized in the nucleus, but does not have a known NLS. In vitro, HMG-1 protein forms complexes with vector DNA. The production of purified HMG-1 is costly and labor-intensive.
Mistry et al. (1997) describe experiments concerning HMG-1-mediated nuclear transport. Due to its positive charge HMG-1, as a transfection reagent that complexes DNA, is used here for the passage of DNA through the cell membrane. The efficiency is low. The company Wako BioProducts (Richmond, Va., U.S.A.) sold (1997) the proteins HMG-1 and -2 as additives for lipofection reagents to mediate nuclear transport.
Fritz et al. (1996) followed a similar approach with calf thymus histones or a recombinant protein consisting of SV40 NLS and human histone H1. Both of these proteins evidently form large complexes with DNA, as was shown in the publication, and are suitable for the passage of the cellular membrane but not for nuclear transport.
B) Due to Their Simpler and Less Expensive Production, Synthetic Peptides Containing NLS Sequences Were Used as Well
The group of P. Aleström (Collas et al., 1996, Collas and Alestrom, 1996, 1997a, b) uses the NLS peptide from SV40 to complex DNA and have it transported into the nucleus by the cell. This DNA binding occurs solely through the positively charged amino acids of the NLS that are essential for its function. This results in masking the actual signal for the nuclear transport proteins as long as the DNA is complexed with the peptide. An NLS-dependent transport of fluorescently labeled DNA could be observed in isolated male pronuclei formed in vitro from sea urchin sperms, when they have been incubated in the lysate of fertilized zebra fish eggs. At a molecular ratio of ≧100:1 (NLS peptide:vector) and ≧1,000 vector copies per cell, an increase in luciferase expression could be observed in zebra fish embryos, when vector DNA was micro-injected. into the cytoplasm of the cells. (At 100 peptides/vector and 1,000 injected vectors a sixfold increase was obtained as compared to 0 peptide.) Due to the high density, possibly not all NLS bind completely to the DNA and thus parts remain accessible for the transport machinery; this might be the cause why an effect can be perceived at all (cf. Sebastyén et al., 1998). The transport machinery is probably able to recognize signals composed of two peptide sequences (Boulikas, 1993).
Sebastyén et al. (1998) covalently coupled many hundreds of SV40 NLS peptides to DNA molecules, with the NLS being scattered over the entire length of the DNA strand. Due to its massive modification, the DNA can no longer be transcribed. As is discussed in the article, the DNA is evidently only transported into the nucleus when so many NLS peptides are bound that, for steric reasons, not all of them are masked by the interaction with the negative charges of the DNA.
Gopal (U.S. Pat. No. 5,670,347) describes a peptide that consists of a DNA-binding basic region, a flexible hinge region and an NLS. As DNA binding is also in this case achieved by the amino acids' positive charges, the reagent forms complexes with the DNA that are meant to serve at the same time for the transport across the cellular membrane. It is not evident why the NLS sequen
Christine Rainer
Siebenkotten Gregor
Mandel & Adriano
Pappu Sita
Yucel Remy
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