Adenoviral vector and methods for making and using the same

Details Adenoviral vector and methods for making and using the same Adenoviral vector and methods for making and using the same

1999-10-27

2001-10-16

Stucker, Jeffrey (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C424S093200, C424S093600, C435S005000, C435S069100, C435S091400, C435S173300, C435S252300, C514S04400A, C536S023720

Reexamination Certificate

active

06303362

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The field of this invention is nucleic acid vectors, particularly adenoviral based vectors.
2. Background of the Invention
The introduction of an exogenous nucleic acid sequence (e.g. DNA) into a cell, a process known as “transformation,” plays a major role in a variety of biotechnology and related applications, including research, synthetic and therapeutic applications. Research applications in which transformation plays a critical role include the production of transgenic cells and animals. Synthetic applications in which transformation plays a critical role include the production of peptides and proteins. Therapeutic applications in which transformation plays a key role include gene therapy applications. Because of the prevalent role transformation plays in the above and other applications, a variety of different transformation protocols have been developed.
In many transformation applications, it is desirable to introduce the exogenous DNA in a manner such that it is incorporated into a target cell's genome. One means of providing for genome integration is to employ a vector that is capable of homologous recombination. Techniques that rely on homologous recombination can be disadvantageous in that the necessary homologies may not always exist; the recombination events may be slow, etc. As such, homologous recombination based protocols are not entirely satisfactory.
Accordingly, alternative viral based transformation protocols have been developed, in which a viral vector is employed to introduce exogenous DNA into a cell and then subsequently integrate the introduced DNA into the target cell's genome. Viral based vectors finding use include retroviral vectors, e.g. Maloney murine leukemia viral based vectors. Other viral based vectors that find use include, HSV derived vectors, sindbis derived vectors, etc. One type of viral vector of particular interest is the adenovirus derived vector.
Recombinant adenovirus vectors have been shown to have great promise for the gene transfer in basic research as well as clinical treatment of many diseases.They can transduce foreign genes efficiently into both cultured cells and many target organs in vivo. There are more forty different serotypes of adenovirus (Ad) identified. The Ad type 5 genome has been most commonly used to make recombinant Ad vector. The genome of human Ad is a linear 36 kb, double-stranded DNA genome that encodes more than 50 gene products. In the first generation Ad vector, the early region 1 (E1) is replaced by the foreign gene and the virus propagated in an E1-transcomplementing cell line such as 293. By deleting E1 and early region 3 (E3) sequences up to about 8 kb of foreign gene can be inserted. However, in vitro manipulation of Ad DNA is difficult. Unique and useful restriction sites are limited because of the large size of the genome, making the construction of Ad vector relatively labor intensive. Two standard methods to make E1-deleted Ad vector have been developed: an in vitro ligation method and a homologous recombination method in 293 cells.
The in vitro ligation method uses whole viral DNA genomes and the plasmid containing the left end of Ad with the right inverted terminal repeat (ITR), the packaging signal and E1A enhancer sequence (map unit; 0 to 1.3). After the gene of interest is inserted into the downstream of the viral sequence of the plasmid, the fragment containing viral sequence and gene of interest is excised and ligated to the unique ClaI site (map unit; 2.6), replacing a portion of the viral E1 region. Then, the ligated DNA is directly transfected into 293 cells to make recombinant virus. However, this method is rarely used today because the efficiency is low and the recombinant virus requires purification of contaminating wild type and transgene null viruses related to incomplete restriction digestion and self-religation.
One system using homologous recombination uses two plasmids with overlapping fragments that recombine in vivo. The first plasmid contains the entire Ad genome with a deletion of the DNA packaging and E1 region. The second plasmid contains right ITR, packaging signal and overlapping sequence with the first plasmid. After the gene of interest is introduced into the second plasmid, the two plasmids are co-transfected into 293 cells. The virus, which is produced by the recombination in 293 cells, is isolated through plaque purification. The major limitation to this approach is that the recombination event occurs at a low frequency.
Newer methods for adenoviral preparation are based on homologous recombination of two plasmids using yeast artificial chromosomes or bacteria. These methods, while more efficient, are more complex. The YAC system requires yeast culture and manipulation while the
E.coli
system requires three step transformations using an additional non-convential host bacterial strain (BJ5183recBCsbcBC).
Accordingly, there is continued interest in the development of new methods for preparing recombinant adenoviral vectors. Of particular interest would be the development of a method which is highly efficient and yet requires a minimum number of steps.
Relevant Literature
U.S. Patents of interest include: U.S. Pat. Nos. 5,962,313; 5,962,311; 5,952,221; 5,932,210; 5,928,944; 5,922,576; 5,919,676; 5,891,690; 5,885,808; 5,880,102; 5,877,011; 5,871,982; 5,869,037; 5,858,351; 5,851,806; 5,843,742; 5,837,484; 5,820,868; 5,789,390; 5,756,283; 5,747,072; 5,731,172; 5,700,470; 5,670,488; 5,616,326; 5,589,377; 5,585,362; and 5,354,678.
Other references of interest include: Berkner, et al., (1983). Generation of adenovirus by transfection of plasmids. Nucleic Acids Res. 11, 6003-6020; Bett, et al. (1994). An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A. 91, 8802-6; Chartier, et al. (1996). Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol. 70, 4805-4810; Crouzet, et al. (1997). Recombinational construction in Escherichia coli of infectious adenoviral genomes. Proc Natl Acad Sci U S A. 94, 1414-1419; Gilardi et al. (1990). Expression of human alpha 1-antitrypsin using a recombinant adenovirus vector. FEBS Lett. 267, 60-2; He, et al. (1998). A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A. 95, 2509-2514; Ketner, et al. (1994). Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone. Proc Natl Acad Sci U S A. 91, 6186-6190; Miyake, et al. (1996). Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci U S A. 93, 1320-1324; and Rosenfeld, et al. (1991). Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science. 252, 431-4.
SUMMARY OF THE INVENTION
In vitro methods for making a recombinant adenovirus genome, as well as kits for practicing the same and the recombinant adenovirus vectors produced thereby, are provided. In the subject methods, the subject genomes are prepared from first and second vectors. The first vector includes an adenoviral genome having an E region deletion and three different, non-adenoviral restriction endonuclease sites located in the E region. The second vector is a shuttle vector and includes a non adenoviral nucleic acid (which is desired to be inserted into the adenoviral genome, i.e. an insertion nucleic acid) flanked by two of the three different non-adenoviral restriction endonuclease sites present in the first vector. Cleavage products are prepared from the first and second vectors using the appropriate restriction endonucleases. The resultant cleavage products are then ligated to produce the subject recombinant adenovirus genome. The subject adenoviral genomes find use in a variety of applications, including as vectors for use in a variety of applications, e.g. gene therapy.


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