Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2000-02-02
2001-10-16
Guzo, David (Department: 1636)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093100, C424S093600, C435S320100, C435S235100, C435S091100, C435S091400, C435S091330, C435S455000, C435S456000
Reexamination Certificate
active
06303116
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to the field of virology, and, more particularly, to a system of generating and producing retroviral vector particles from retroviruses which are non-pathogenic for humans and capable of transducing therapeutic genes into non-dividing cells.
BACKGROUND OF THE INVENTION
All current retroviral vectors used in human gene therapy protocols have been derived from murine leukemia virus (MLV), an amphotropic C-type retrovirus. However, like all other C-type retroviruses, MLV can only establish an infection in the target cell after one cell division. To bypass this problem, efforts are underway in several laboratories to develop retroviral vectors from lentiviruses, such as the human immunodeficiency virus (HIV-1) the major causative agent of AIDS, which are capable of infecting non-dividing cells. However, serious safety issues make it highly unlikely that such vectors could be used in the clinic.
The present invention describes procedures for the generation and production of safe retroviral vectors derived from C-type retroviruses, which are non-pathogenic for humans and which are capable of transducing genes into specific non-dividing cells. Transduction of genes into non-dividing cells has been achieved in the present invention by using the approaches described herein. Using site-directed mutagenesis, a specific signal (nuclear transportation signal sequence) has been introduced into the matrix protein of the retroviral vector particle. This signal is sufficient to enable the penetration of the nucleus of the non-dividing cell.
The introduction of a nuclear localization sequence into the matrix protein adds a new feature to the C-type retroviral vector particle: the capability to actively penetrate the nucleus of a quiescent cell. This system has two major advantages over current vector systems. First, efficient gene transfer into non-dividing cells is achieved. Moreover, this system is, but does not have to be, combined with an existing cell-type-specific gene delivery system. Second, this system is safe, since the retroviral particles used have been derived from a retrovirus which is non-pathogenic in humans.
DEFINITIONS
“Nuclear translocation sequence” means nuclear localiation sequence or nuclear transportation signal sequence.
SUMMARY OF THE INVENTION
The retroviral vector system described here may be used to deliver genes into various tissues of the human body, which consists of non-dividing cells, e.g., liver, hematopoietic stem cells, brain and many more cell-types. It can be combined with a cell-type-specific gene delivery system developed by us earlier to transfer genes into a very distinctive cell-type only. Thus, there will be numerous human gene therapy applications into various organs transducing a large variety of therapeutic genes. The system described here overcomes the last major hurdle and disadvantage of current retroviral vector systems, which is the incapability to infect non-dividing cells.
This invention also relates to a method for preparing particles, which contain genetically modified core proteins involved in the import of retroviral cores into the nucleus.
The present invention describes procedures for the generation and production of safe retroviral vectors derived from retroviruses, which are non-pathogenic for humans and capable of transducing genes into non-dividing cells. This has been achieved using the following approaches. Using site-directed mutagenesis, a specific signal (nuclear transportation signal sequence); FIG.
2
and SEQ. ID. NO: 1-10 has been introduced into the core protein of the retroviral vector particle. This signal is sufficient to enable the penetration of the nucleus of the non-dividing cell.
In one embodiment, the present invention pertains to a C-type retroviral vector particle having a genetically modified MA core protein, wherein a consensus nuclear translocation sequence (HIV-1 census sequence, SEQ. ID. NO: 11) has been created by altering the amino acid sequence of the wild-type core protein.
In another embodiment, the present invention pertains to a method for preparing a C-type retroviral particle having the capability to infect quiescent cells which comprises a retroviral vector core particle which contains a genetically engineered MA protein wherein a nuclear translocation sequence has been created by altering the amino acid sequence of the wild-type MA protein.
In yet another embodiment, the present invention pertains to a C-type retroviral particle having the capability to infect quiescent cells which comprises a retroviral vector core particle which contains a genetically engineered MA protein, wherein a nuclear translocation sequence has been created by altering the amino acid sequence of the wild-type MA protein, which facilitates nucleus penetration of the infected target cell.
In yet another embodiment, the present invention pertains to a method for preparing a C-type retroviral particle having the capability to infect quiescent cells which comprises a retroviral vector core particle which contains a genetically engineered MA protein wherein a nuclear translocation sequence has been created by altering the amino acid sequence of the wild-type MA protein, which facilitates nucleus penetration of the infected target cell.
REFERENCES:
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Guzo David
Smith Janet B.
Thomas Jefferson University
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