The present invention relates to a viral vector for the gene therapy. More specifically, this invention relates to a negative strand RNA viral vector.
As to the gene therapy for humans and animals, therapeutic effectiveness and safety are very important factors. Especially, therapy performed by using xe2x80x9cviral vectorxe2x80x9d 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 xe2x80x9cinfectivityxe2x80x9d in this specification is meant the xe2x80x9ccapability 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 membranexe2x80x9d. 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 xe2x80x9cdisseminative capabilityxe2x80x9d is meant xe2x80x9cthe 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 acidxe2x80x9d. 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 the transcription promotor, resulting in the expression of the desired products from the foreign gene within cells. Furthermore, the present inventors succeeded in forming an infectious but not disseminative complex by the co-presence of cDNA unit (helper) for expressing the viral structural gene and that for expressing said RNA vector in the packaging cells.
Positive strand RNA viral vectors are expected to be useful as RNA vectors with autonomous replicating capability, but their use as vectors for gene therapy poses-the following problems.
1. Since they are of the icosohedral structure, the size of foreign gene allowed to be inserted is limited to 3,700 nucleotides at most.
2. Until nucleic acids are released from the packaged complex into the cell and replicated, as many as five processes are required, including cellular adhesion, endocytosis, membrane fusion, decapsidation and translation of replication enzymes.
3. A possible formation of disseminative viral particles even in a minute quantity during packaging cannot be denied. Especially, even with attenuated viral particles, the inside RNA itself has disseminative potency and may belatedly be amplified, making it difficult to check.
4. Since these vectors are derived from viruses transmitted to animals by insects such as mosquitoes, when animals and humans to which such vector genes are transferred and are mix-infected with wild type viruses, disseminative recombinants may be formed, possibly further creating a risk of said recombinants being scattered to the natural environment by insects.
Such problems described above are conceived to be basically overcome if RNA viral vectors derived from negative strand RNA viruses are constructed. That is, since negative strand RNA viruses do not have the capsid of icosohedral structure but have a helical nucleocapsid, and also since the envelope size of particles is known to vary depending on the inside RNA content, they are supposed to be much less restricted, compared with positive strand RNA viruses, with respect to the size of foreign genes to be inserted when used as RNA viral vectors. Further, since a group of proteins required for transcription and replication are packaged into particles, only two processes are required, including cellular adhesion and membrane fusion, until nucleic acids are replicated. Furthermore, viral RNA alone is not disseminative. In addition, most of negative strand RNA viruses are not transmitted by insects.
In spite of many advantages of negative strand RNA viruses which may be used as the source of industrially useful viral vectors, no negative strand RNA vectors applicable for gene therapy has become available until now. This is probably due to tremendous difficulties in re-constituting viral particles via viral cDNA. Since the gene manipulation on the DNA level is required to insert foreign genes into vectors, so far as viral particles are not reconstructed from viral cDNA with a foreign gene inserted, it is difficult to use negative strand RNA viruses as a vector. xe2x80x9cReconstruction of viral particlesxe2x80x9d refers to the formation of the original virus or a recombinant virus in vitro or intracellularly from artificially prepared cDNA encoding the viral RNA genome.
As described above, it has been clearly demonstrated that, even if the viral RNA (vRNA) of negative strand RNA viruses or its complementary strand RNA (cRNA; complementary RNA) alone is transferred into cells, no progeny virus can be generated. This is a definitely different point from the case of positive strand RNA viruses, whose RNA can initiate viral life cycle and generate progeny viruses, when transferred into cells. Although, in JP-A-Hei 4-211377, xe2x80x9cmethods for preparing cDNA corresponding to a nonsegmented negative strand RNA viral genome and infectious negative strand RNA virusxe2x80x9d are described for measles virus, a paramyxovirus, the entire experiments of said document described in xe2x80x9cEMBO. J., 9, 379-384 (1990)xe2x80x9d were later proved to be not reproducible, so that the authors themselves had to withdrew all the article contents [ref. EMBO. J., 10, 3558 (1991)]. Therefore, it is obvious that techniques described in JP-A-Hei 4-211377 for another paramyxovirus, Sendai virus, do not correspond to the related art of the present invention.
With regard to the reconstitution system for negative strand RNA viruses, there are reports on influenza virus [Annu. Rev. Microbiol., 47, 765-790 (1993); Curr. Opin. Genet. Dev., 2, 77-81 (1992)]. Influenza virus is an eight-segmented negative strand RNA virus. According to these literatures, a foreign gene was first inserted to a cDNA corresponding to one of said segments, and the RNA transcribed from the cDNAs is assembled with the virus-derived NP protein and RNA polymerase proteins to form an RNP. Then, cells are transfected with this RNP and superinfected with an appropriate intact influenza virus. A reassortant virus emerges, in which the corresponding segment is replaced with the engineered segment, which can be selected under appropriate pressures. Several years later, virus-reconstitution entirely from cDNA of nonsegmented negative strand RNA virus was reported with rabies virus belonging to rhabdoviruses [EMBO J. 13, 4195-4202 (1994)].
However, it has been difficult to use these virus reconstitution techniques as such for constructing vectors for gene therapy because of the following problems.
1. Reconstituted viruses were identified only by the expression of marker gene, RT-PCR, etc. No re-constitution system for the production of vector viruses in a satisfactory yield has been established.
2. Differing from the case of positive strand RNA viruses, in order to form complexes with infectivity but deficient in disseminative potency as vectors for gene therapy, it is necessary to enclose factors required for primary transcription and replication within the complex. No technique for amplifying these complexes in a large scale has been established.
3. For the purpose of intracellularly providing factors necessary for viral reconstitution, cells to which cDNAs are introduced are mix-infected with helper viruses such as recombinant vaccinia virus, etc. to allow transcription of the plasmids supplying those viral protein factors in trans. It is not easy to separate these natural type viruses from the recomstituted viruses.
Furthermore, as one problem with regard to RNA viral vectors in general, it is conceivably necessary to beforehand provide inhibitors for replication of RNA viral vectors which have no effects on host""s replication and transcription, providing for the case where RNA replicated and transcribed in large amounts exerts undesirable effects on treated humans and animals. However, no such inhibitors have been developed.
Objects of the present invention are to develop negative strand RNA viral vectors for practical use, methods for efficiently preparing said vectors, and inhibitors for the replication of said vectors.
The present inventors first attempted to reconstitute Sendai virus from nucleic acids of said virus which is a typical nonsegmented, negative strand RNA virus, and conceived to be industrially most useful as a vector from the viewpoints of safety and convenience. First, in order to apply to the reconstitution test, various investigations were performed using cDNA encoding a Sendai virus minigenome as experimental materials. A cDNA plasmid was constructed so that the Sendai virus protein coding region of about 14 kb is replaced with a reporter luciferase gene and this construct is flanked by T7 promoter and hepatitis delta virus ribozyme gene. As a result, the inventors found efficient conditions regarding weight ratios among materials to be transferred into host cells, including minigenome cDNA, cDNAs encoding the nucleocapsid protein (N), the large protein (L), and the phosphoprotein (P), and minimizing cytotoxicity induced by the recombinant vaccinia virus to provide the T7RNA polymerase. The N protein encapsidate the naked viral RNA to form the RNP, which is now active as the template for both viral mRNA synthesis and viral replication. Furthermore, the present inventors obtained full-length cDNAs corresponding to both positive and negative strands, constructed plasmids for inducing the intracellular biosynthesis of either positive strand RNA (antigenome or cRNA) or negative strand RNA (genome or vRNA) of Sendai virus, and transferred said plasmids into host cells wherein N, P, and L proteins from the respective cotransfected plasmids were expressed. As a result, the inventors first succeeded in re-constructing Sendai virus particles from cDNAs derived thereof.
That is, the present invention comprises the followings.
1. A complex comprising an RNA molecule derived from a specific disseminative negative strand RNA virus and viral structural components containing no nucleic acids, having the infectivity and autonomous RNA replicating capability, but deficient in the disseminative capability.
2. The complex of description 1, wherein said specific RNA virus is a negative strand RNA virus having non-segmented genome.
3. The complex of description 2, wherein said specific RNA virus is Sendai virus.
4. An RNA molecule comprising Sendai viral RNA (vRNA) or its complementary RNA (cRNA), wherein said RNA molecule is defective in that at least one or more than one gene coding for each of the M, F and HN proteins are deleted or inactivated.
5. A complex comprising the RNA of description 4 and viral structural components containing no nucleic acids derived from Sendai virus, having the infectivity and autonomous RNA replicating capability, but deficient in the disseminative capability.
6. A DNA molecule comprising a template DNA transcribable to the RNA molecule of description 4 in vitro or intracellularly.
7. The complex of any one of descriptions 1-3 or 5, wherein the RNA molecule contained in said complex comprises a foreign gene.
8. The complex of descriptions 3 or 5, wherein the RNA molecule contained in said complex comprises a foreign gene.
9. The RNA molecule of description 4 comprising a foreign gene.
10. The DNA molecule of description 6 comprising a foreign gene.
11. An inhibitor for RNA replication contained in the complex of any one of descriptions 1-3, 5, 7 or 8 comprising an inhibitory drug for the RNA-dependent RNA replication.
12. A host whereto the complex of any one of descriptions 1-3, 5, 7 or 8 can disseminate.
13. The host of description 12 comprising a group of genes related to the infectivity of the complex of any one of descriptions 1-3, 5, 7 or 8 on its chromosomes, and capable of replicating the same copies of said complex when infected with it.
14. The host of descriptions 12 or 13, wherein said host is animals, or cells, tissues, or embryonated eggs derived from it.
15. The host of description 14 wherein said animal is mammalian.
16. The host of description 14 wherein said animal is avian.
17. A host comprising a group of genes related to the infectivity of the complex of any one of descriptions 3, 5 or 8 on its chromosomes, and capable of replicating the same copies of said complex when infected with it.
18. A host comprising at least more than one gene of the M, F and HN genes of Sendai virus or genes having functions equivalent to them on its chromosomes.
19. A host comprising the M, F, or HN gene of Sendai virus or each of their functionally equivalent genes on its chromosomes.
20. A host comprising the M, NP, P and L genes of Sendai virus on its chromosomes (wherein each gene may be substituted with its functionally equivalent gene, respectively).
21. A host comprising the M, F and HN genes of Sendai virus on its chromosomes (wherein each gene may be substituted with its functionally equivalent gene, respectively).
22. A host comprising the M, F, HN, NP, P and L genes of Sendai virus on its chromosomes (wherein each gene may be substituted with its functionally equivalent gene, respectively).
23. The host of any one of descriptions 17-22, wherein said host is animal, or cell, tissue or egg derived from it.
24. The host of description 23, wherein said animal is mammalian.
25. The host of description 23, wherein said animal is avian.
26. A kit consisting of the following three components,
a. the RNA molecule contained in the complex of any one of descriptions 1-3, 5, 7 or 8, or cRNA of said RNA, or a unit capable of biosynthesizing said RNA or said cRNA,
b. a group of enzymes required for replicating said RNA or said cRNA, or a unit capable of biosynthesizing said group of enzymes, and
c. a group of proteins related to the infectivity of said complex, or a unit for biosynthesizing said group of proteins.
27. A kit consisting of the following three components,
a. the RNA molecule contained in the complex of any one of descriptions 1-3, 5, 7 or 8, or cRNA of said RNA, or a unit capable of biosynthesizing said RNA or said cRNA,
b. a group of enzymes required for replicating said RNA or said cRNA, or a unit capable of biosynthesizing said group of enzymes, and
c. the host of any one of descriptions 12-25.
28. A kit consisting of the following two components,
a. the complex of any one of descriptions 1-3, 5, 7 or 8, and
b. the host of any one of descriptions 12-25.
29. A kit consisting of the following three components,
a. the RNA molecule contained in the complex of any one of descriptions 3, 5 or 8, or cRNA of said RNA, or a unit capable of biosynthesizing said RNA or said cRNA,
b. all NP, P and L proteins of Sendai virus, or a unit for biosynthesizing said group of proteins, and
c. a group of proteins related to the infectivity of said complex, or a unit for biosynthesizing said group of proteins.
30. A kit consisting of the following three components,
a. the RNA molecule contained in the complex of any one of descriptions 3, 5 or 8, cRNA of said RNA, or a unit capable of biosynthesizing said RNA or said cRNA,
b. all NP, P and L proteins of Sendai virus, or a unit capable of biosynthesizing said group of proteins, and
c. the host of any one of descriptions 17-25.
31. A kit consisting of the following two components,
a. the complex of any one of descriptions 3, 5 or 8, and
b. the host of any one of descriptions 17-25.
32. A method for producing the complex of any one of descriptions 1-3, 5, 7 or 8 by introducing three components of descriptions 26a, 26b and 26c into a host.
33. A method for producing the complex of any one of descriptions 1-3, 5, 7 or 8 by introducing both components of descriptions 27a and 27b into the host of description 27c. 
34. A method for amplifying and producing the complex of description 28a by transfecting said complex to the host of description 28b. 
35. A method for producing the complex of any one of descriptions 3, 5 or 8 by introducing the three components of descriptions 29a, 29b and 29c into a host.
36. A method for producing the complex of any one of descriptions 3, 5 or 8 by introducing both components of descriptions 30a and 30b into the host of description 30c. 
37. A method for amplifying and producing the complex of description 31a by transfecting said complex into the host of description 31b. 
38. The RNA molecule of description 9 wherein a gene corresponding to the M, F, or HN gene is deleted or inactivated.
39. The RNA molecule of description 9 wherein all the genes corresponding to the M, F and HN genes are deleted or inactivated.
40. A kit consisting of the following three components,
a. the RNA molecule of description 38,
b. the host of description 20, and
c. the host of description 19.
41. A method for producing a complex by introducing the RNA molecule of description 40a into the host of description 40b, and amplifying and producing said complex by transfecting it into the host of description 40c. 
42. A complex produced by the method of description 41.
43. A kit consisting of the following three components,
a. the RNA molecule of description 39,
b. the host of description 22, and
c. the host of description 21.
44. A method for producing a complex by introducing the RNA molecule of description 43a into the host of description 43b, and amplifying and producing said complex by transfecting it into the host of description 43c. 
45. A complex produced by the method of description 44.
46. An inhibitor for RNA replication contained in the complex of either descriptions 42 or 45 comprising an inhibitory drug of the RNA-dependent RNA replication.
47. A method for preparing the foreign proteins, wherein said method comprises the process of introducing the complex of description 7 to a host and the process of recovering the expressed foreign proteins.
48. A method for preparing the foreign proteins of description 47, wherein the host is a cell expressing a group of genes, from among those related to the disseminative capability, which are deficient in the RNA molecule contained in the complex of description 7.
49. A culture medium or allantoic fluid containing the expressed foreign proteins, wherein said culture medium or allantoic fluid is obtained by inoculating the complex of description 7 into a host and recovering it.