Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-12-23
2003-11-11
Shukla, Ram (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S320100, C435S069100, C536S023100
Reexamination Certificate
active
06645760
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a viral vector for the gene therapy. More specifically, this invention relates to a negative strand RNA viral vector.
BACKGROUND OF THE INVENTION
As to the gene therapy for humans and animals, therapeutic effectiveness and safety are very important factors. Especially, therapy performed by using “viral vector” expressing a foreign gene of concern which is obtained by gene recombination of the viral genome and the foreign gene needs to be very cautiously carried out, when such undeniable possibilities exist as that the recombinant virus may be inserted to unspecified sites of chromosomal DNA, that the recombinant virus and pathogenic virus may be released to the natural environment, and that the expression level of gene transfected into cells cannot be controlled, or the like, even though its therapeutic effectiveness is recognized.
These days, a great number of gene therapies using recombinant viruses are performed, and many clinical protocols of gene therapy are proposed. Characteristics of these recombinant viral vectors largely depend on those of the viruses from which said vectors are derived.
The basic principle of viral vector is a method for transferring the desired gene into targeted cells by utilizing the viral infectivity. By “infectivity” in this specification is meant the “capability of a virus to transfer its nucleic acid, etc. into cells through its adhesiveness to cells and penetrating capability into cells via various mechanisms including fusion of the viral membrane and host cellular membrane”. With the surface of recombinant viral vectors genetically manipulated to insert a desired gene are associated the nucleocapsid and envelope proteins, etc. which are derived from the parental virus and confer infectivity on the recombinant virus. These proteins enable the transfer of the enclosed recombinant gene into cells. Such recombinant viral vectors can be used for the purpose of not only gene therapy, but also production of cells expressing a desired gene as well as transgenic animals.
Viral vectors are classified into three classes comprising the retroviral vector, DNA viral vector and RNA viral vector.
These days, the vectors most frequently used in gene therapy are retroviral vectors derived from retroviruses. Retroviruses replicate through the following processes. First, upon penetration into cells, they generate complementary DNAs (cDNAs) using their own reverse transcriptase as at least part of catalysts and their own RNA templates. After several steps, said cDNAs are incorporated into host chromosomal DNAs, becoming the proviruses. Proviruses are transcribed by the DNA-dependent RNA polymerase derived from the host, generating viral RNAS, which is packaged by the gene products (proteins) translated from the RNAS. The RNAs and proteins finally assemble to form mature virus particles.
In general, retroviral vectors used in gene therapy, etc. are capable of carrying out processes up to provirus generation. However, they are deficient viruses deprived of genes necessary for their packaging of the progeny genome RNA so that they do not form viral particles from provirus. Retroviruses are exemplified by, for example, mouse leukemia virus, feline leukemia virus, baboon type C oncovirus, human immunodeficiency virus, adult T cell leukemia virus, etc. Furthermore, recombinant retroviral vectors hitherto reported include those derived from mouse leukemia virus [see Virology, 65, 1202 (1991), Biotechniques, 9, 980 (1989), Nucleic Acids Research, 18, 3587 (1990), Molecular and Cellular Biology, 7, 887 (1987), Proceedings of National Academy of Sciences of United States of America, 90, 3539 (1993), Proceedings of National Academy of Sciences of United States of America, 86, 3519 (1989), etc.] and those derived from human immunodeficiency virus [see Journal of Clinical Investigation, 88, 1043 (1991)], etc.
Retroviral vectors are produced aiming at efficiently inserting a desired specific DNA into the cellular chromosomal DNA. However, since the insertion position of the DNA is unpredictable, there is undeniable possibilities such as the damage of normal genes, activation of oncogene and excessive or suppressive expression of desired gene, depending the position of insertion. In order to solve these problems, a transient expression system using DNA viral vectors which can be used as extrachromosomal genes has been developed.
DNA viral vectors are derived from DNA viruses, having DNA as genetic information within viral particles. Replication of said DNA is carried out by repeating the process of generating complementary DNA strand using DNA-dependent DNA replicase derived from host as at least one of catalysts with its own DNA as template. The actual gene therapy using adenoviral vector, a DNA viral vector usable as extrachromosomal gene, is exemplified by the article in [Nature Genetics, 3, 1-2 (1993)]. However, since, in the case of DNA viral vectors, the occurrence of their undesirable recombination with chromosomal DNA within nucleus is also highly possible, they should be very carefully applied as vectors for gene therapy.
Recently, RNA viral vectors based on RNA viruses have been developed as conceivably more safer vectors than DNA and retroviral vectors described above. RNA viruses replicate by repeating the processes for generating complementary strands using their own RNA-dependent RNA replicase as the catalyst with their own RNA as template.
The genome RNA of positive strand RNA viruses have dual functions as the messenger RNA (hereafter simply called mRNA), which generate proteins, depending on the translational functions of host cells, necessary for the replication and viral particle formation and as the template for genome replication. In other words, the genome RNA itself of positive strand RNA viruses has a disseminative capability. In the present specification, by “disseminative capability” is meant “the capability to form infectious particles or their equivalent complexes and disseminate them to other cells following the transfer of nucleic acid into host cells by infection or artificial techniques and the intracellular replication of said nucleic acid”. Sindbis virus classified to positive strand RNA viruses and Sendai virus classified to negative strand RNA viruses have both infectivity and disseminative capability. Adeno-satellite virus classified in Parboviruses is infectious but not disseminative (mixed infection with adenovirus is required for the formation of viral particles.). Furthermore, the positive strand RNA derived from Sindbis virus which is artificially transcribed in vitro is disseminative (forming infectious viral particles when transfected into cells), but neither positive nor negative RNA strands of Sendai virus artificially transcribed in vitro is disseminative, generating no infectious viral particles when transfected into cells.
In view of the advantage that the genome RNA functions as mRNA at the same time, the development of RNA viral vectors derived from positive strand RNA viruses preceded [see Bio/Technology, 11, 916-920 (1993), Nucleic Acids Research, 23, 1495-1501 (1995), Human Gene Therapy, 6, 1161-1167 (1995), Methods in Cell Biology, 43, 43-53 (1994), Methods in Cell Biology, 43, 55-78 (1994)]. For example, RNA viral vectors derived from Semliki forest virus (SFV) [Bio/Technology, 11, 916-920 (1993)] and Sindbis virus are basically of the RNA structure wherein the structural protein gene regions related to the viral structure are deleted, and a group of genes encoding proteins necessary for viral transcription and replication are retained with a desired foreign gene being linked downstream of the transcription promotor. Direct transfer of such recombinant RNA or cDNA which can transcribe said RNA [Nucleic Acids Research, 23, 1495-1501 (1995)] into cells by microinjection, etc. allows autonomous replication of RNA vectors containing the foreign gene, and the transcription of foreign gene inserted downstream of
Asakawa Makoto
Hasegawa Mamoru
Kato Atsushi
Murai Fukashi
Nagai Yoshiyuki
Dnavec Research Inc.
Shukla Ram
Smith Chalin A.
Smith Patent Consulting
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