Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2000-10-12
2004-09-28
Foley, Shanon (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S236000, C435S320100, C536S023720
Reexamination Certificate
active
06797506
ABSTRACT:
The present invention relates to viral vectors and viral DNA.
Adenovirus has been studied for its role in human disease (25), as a model for many important discoveries in molecular biology, including mRNA splicing, DNA replication, transcription and cell transformation (reviewed in 44) and more recently as a powerful reagent for transient gene expression (12, 46). A detailed understanding of the adenoviral life cycle is well established (reviewed in 50). Since the initial efforts to use adenovirus as a gene transfer vector (18, 52, 28) the virus has gained in popularity as a vector and a number of methods of generating alterations in the viral genome to carry novel genes have been developed (2, 5, 11, 15, 21, 23, 26, 32, 38, 41, 43, 48 reviewed in 31, 49). Because of the ease of vector construction and purification, and because these vectors have a potent ability to transiently transduce novel genetic material into a variety of mammalian cell types in vivo, adenovirus vectors were used extensively in early efforts at clinical gene therapy.
Unfortunately, several features of the adenovirus type 5 (Ad5) based vectors initially used have limited the success in the initial applications. These included both the host immune response to adenovirus (reviewed in ref. 55) as well as the failure of the virus to efficiently enter certain target cell types (20, 58, 59). Thus, there is now an interest in adenovirus types that could provoke less aggressive host immune responses and could enter target cells with greater efficiency.
A large number of alternate adenovirus serotypes are known and may provide advantages in some applications over Ad5-based vectors. Additional adenoviruses that have recently been modified as vectors include the ovine adenovirus 287 (29, 53, 56,), the bovine adenovirus type 3 (40, 60), and the canine adenovirus (30). It was considered that these alternate serotypes would provide both a novel vector backbone to which there is no pre-existing immune response in the target host. Furthermore, because adenoviruses are extremely species specific in their replication capacity (50) a degree of security against inappropriate vector replication is gained by using an vector derived from a distant species of adenovirus.
There are several justifications for pursuing these alternate viral subtypes. For vaccine applications in their non-human hosts, these viruses, if properly modified, may provoke more effective immune responses than a human adenovirus based vector. Furthermore, more robust immune responses might be expected from a replication competent virus; thus a vector is most useful in a host where replication is partially or fully permissive. This is not the case with human adenovirus based vectors in nearly all nonhuman hosts.
It has been an object of the invention to provide an alternative adenovirus vector for use as a gene delivery vector and for use as a vaccine.
To solve the problem underlying the present invention, the avian adenovirus CELO has been chosen to be modified. CELO (chicken embryo lethal orphan or fowl adenovirus type 1, reviewed in 39) was characterized as an infectious agent in 1957 (57). There are few serious health or economic consequences of CELO virus infection. CELO can be isolated from healthy chickens and in general, do not cause disease when experimentally re-introduced into chickens (10).
CELO virus is structurally similar to the mammalian adenoviruses (mastadenoviruses) with an icosahedral capsid of 70-80 nm made up of hexon and penton structures (33); the CELO virus genome is a linear, double-stranded DNA molecule with the DNA condensed within the virion by virus-encoded core proteins (33, 36). CELO virus has a larger genome than Ad5 (44 kb vs. ca. 36 kb, ref. 6, WO 97/40180). The CELO virion has two fibers of different lengths at each vertex (24, 33, 35) rather than the single fiber of most other serotypes (reviewed in 50). The CELO virus is not able to complement the E1A functions of Ad5 and CELO virus replication is not facilitated by Ad5 E1 activity (37). The complete DNA sequence of CELO (6, WO 97/40180) revealed additional differences between CELO virus and the mastadenoviruses including the absence of sequences corresponding to the Ad5 early regions E1A, E1B, E3 and E4. The CELO genome contains approximately 5 kb of sequence at the left end and 12 kb at the right end, rich in open reading frames, which have no sequence homology to Ad5 but probably encode the early functions of the virus.
When developing CELO into a gene delivery vector, it has been considered that the virus is naturally defective in mammalian cells and this property should limit the possibility of complementation by wildtype mammalian adenovirus. The CELO virion has increased DNA packaging capacity and much greater physical stability than the virion of Ad5. One practical feature of CELO is the ability to grow the virus in chicken embryos, a system of low cost and high convenience (9, 33).
For application in avian systems, especially for vaccine applications, it would be useful to have a CELO derivative with reduced replication capacity. Such a replication defective virus would allow transduction of avian species or avian cells similar to replication competent CELO vectors, but the amplification and spread of the modified virus would be limited by the impaired replication capacity of the virus.
A gene termed Gam1 was originally identified in the CELO genome in a search for viral genes that influence cell survival (7). The Gam1 protein is encoded by CELO nucleotides 37391-38239, transcriptional control sequences are located within ca. 1500 basepairs upstream and ca. 300 basepairs downstream of the coding region.
The present invention relates to recombinant CELO virus or CELO virus DNA that have the region spanning nucleotides 37391-39239 of the CELO wild type virus genome completely or partially deleted or altered or that contain an insertion in this region, any of which modifications results in a complete loss of Gam1 expression or prevents the expression of a functional Gam1 protein. Alternately to a disruption in the region defined above, the virus may contain a disruption in the transcriptional control elements of the Gam1 gene. (For simplicity, any modification that results in a complete loss of Gam1 expression or an inhibition of functional Gam1 expression will be referred to “Gam1 disruption” in the following.)
The suitability of a CELO mutation for obtaining a virus of the invention can be readily determined by ascertaining the absence of Gam1 expression. Suitable tests employ standard immunological methods, e.g. immunofluorescence microscopy or Western immunoblotting using antisera specific for the Gam1 protein.
The CELO nucleotide sequence numbering used in the present invention is derived from reference 6, WO 97/40180 and GenBank U46933, which describe the sequence of the wild type CELO virus genome.
CELO virus or CELO virus DNA with Gam1 disruptions, and their derivatives respectively, have been designated CELO AIM65 or CELO AIM65 derivatives, respectively.
In an embodiment, the invention is directed to a CELO AIM65 derivative with a complete or partial deletion and/or insertion within the Gam1 gene as defined above.
Preferably, the nucleotides 37391-39239 are completely deleted to provide more space for inserting the foreign DNA in place of the deletion.
In another preferred embodiment, the deletion comprises the region defined by the rescriction sites SmaI/Bgl II, i.e. the region spanning nt 36818-37972, which removes part of the Gam1 open reading frame.
The deletions defining CELO AIM65 may be combined with mutations defining CELO AIM46 (79) or its derivatives. Examples for AIM46 mutations are complete or partial deletion(s) of the regions spanning nt 41731-43684, 41523-43684, 41002-43684 or 40065-43684. Besides, the CELO AIM65 modifications may also be combined with the modifications described for AIM69 and/or AIM70 (79).
Thus, the CELO virus and CELO virus DNA of the invention carry a Gam1 disruption that is optionally combined with a deletion spanning
Cotten Matthew
Glotzer Jolanta
Michou Anne-Isabelle
Boehringer Ingelheim International GmbH
Foley Shanon
Sterne Kessler Goldstein & Fox PLLC
LandOfFree
Recombinant, replication defective CELO virus and CELO virus... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant, replication defective CELO virus and CELO virus..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant, replication defective CELO virus and CELO virus... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3242105