Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1999-07-28
2002-09-17
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S440000, C435S455000, C435S006120, C435S320100, C435S069100, C435S015000, C424S900000, C514S04400A, C530S350000, C530S416000, C530S333000
Reexamination Certificate
active
06451579
ABSTRACT:
BACKGROUND AND SUMMARY OF THE INVENTION
Alphaviruses are single-stranded RNA viruses with a spike protein envelope structure. Even though infection usually occurs via a receptor-mediated endocytosis, naked viral RNA can initiate infection when introduced into the cytoplasm of a wide variety of host cells, including vertebrate and invertebrate cells. The 5′ two-thirds of the 12-kb viral RNA encodes the four viral nonstructural, or replicase proteins nsp 1, nsp 2, nsp 3 and nsp 4, required for RNA amplification in the infected cell. The remaining third of the viral RNA codes for the structural proteins, the viral capsid and spike proteins, which are translated from the subgenomic 26S RNA with its own 26S promoter. Once the positive-sense RNA genome of the alphavirus is in the cytoplasm, it serves as the template for synthesis of a complementary negative strand by the virus-encoded replicase. The negative strand serves as the template for additional genomic RNA. Viral RNA replication is extremely rapid resulting in packaging of high titer alphavirus stocks of up to 10
10
units per milliliter.
Due to its high replication efficiency, the simplicity of the replication cycle, i.e. the ability of the virus to replicate without a DNA intermediate, and its wide host range, alphaviruses have been studied for their potential as virus-based expression vectors. Since the virus requires only the presence of the replicase to replicate, the region encoding the replicase and all sequences required in cis for replication and packaging are maintained and the region encoding the structural proteins can be replaced with a desired gene. This type of viral vector with a replication capability is termed a replicon. It is currently possible with the alphavirus expression systems to split the replicon into two distinct replicons (Liljestrom and Garoff (1991)
Bio/Technology
9:1356-1361; Xiong et. al. (1989)
Science
243:1188-1191). One replicon contains all the viral structural genes and a replication signal, and is known as a helper plasmid or RNA. The second replicon contains the nonstructural proteins coding sequences, a signal for packaging of the RNA replicon, an expression cassette for expression of foreign genes (under control of the 26S promoter), and is known as the expression replicon. When the two RNA replicons are transfected into cells, it is possible for the nonstructural proteins of the expression vector to replicate the helper RNA and express the structural proteins, thus allowing for specific packaging of the expression replicon. These viral particles can then be used to infect cells and express protein. The replication of these RNA replicons is very rapid and a single RNA molecule can replicate up to 100,000 copies or more in 4-6 hours (Wengler, G. (1980) In:
The Togaviruses
. R. W. Schlesinger (ed.), Academic Press, New York, pp. 459-472).
The alphavirus expression systems using Semliki forest virus (SFV) (Liljestrom and Garoff (1991)
Bio/Technology
9:1356-1361) and Sindbis (Xiong et al. (1989)
Science
243:1188-1191; Dubensky et al. (1996)
J. Virol.
70:508-519) have been extensively used to express recombinant proteins transiently, either as a transient transfection system, or by using viral particles to infect cells (Lundstrom K. (1997)
Cur. Opin. Biotechnol.
8:578-582; Ciccarone et al. (1994)
Focus
15: 103-105; Liljestrom, P. (1994)
Curr. Opin. Biotechnol.
5:495-500). These systems have several advantages over other expression systems in that: 1) expression levels are high, and 2) viral particles can be generated and used to infect a variety of cell types so that authentic, post-translationally modified proteins can be made (Lundstrom et al. (1997)
Eur. J. Pharmacol.
337:73-81).
Due to the fact that SFV and Sindbis are human pathogens, there are disadvantages with the viral expression system because of biosafety issues and problems with scale up. Under current regulations, the use of >10
9
viral particles requires a higher level of containment than a Biosafety Level 2 (BL2) laboratory making large scale viral infection and production, and subsequently, large scale manufacturing of proteins, problematic. Additionally, the viral life cycle is lethal in that a viral infection will kill cells at 48-96 hours post-infection or transfection, regardless of whether the viral RNA is introduced as packaged viral particles or by transfection of RNA followed by replication and protein expression. Some approaches have been described to control the replication rate of alphavirus RNA such as the overexpression of the anti-apoptotic protooncogene bcl-2 (Scallan, M. F. et al. (1997)
J. Virol.
71:1583-1590), but this strategy only temporarily slowed viral replication and host cell apoptosis.
Therefore, there is a need for a non-viral or viral based expression system in which one could control the replication rate of these replicons so that they are not lethal to the host cell and which would allow for large-scale applications. In this system, an alphavirus DNA vector is utilized.
Alphavirus DNA vectors have been developed for both SFV (Berglund P., Tubulekas I., Liljeström P., Alphavirus as vectors for gene delivery,
Trends Biotechnol
1996, 14:130-134) and Sinbis (Johanning F. W., Conry R. M., LoBuglio A. F., Wright M., Sumerel L. A., Pike M. J., Curiel D. T., A Sinbis Virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo,
Nucleic Acids Res
1995, 23:1495-1501; Herweijer H., Latendresse J. S., Williams P., Zhang G., Danko I., Schlesinger S., Wolf J. A., A plasmid-based self-amplifying Sindbis virus vector,
Hum Gene Ther.
1995, 6: 1161-1167). In these DNA-RNA layered vectors a eukaryotic promoter is introduced upstream of the alphavirus replicase genes. DNA is transcribed to RNA from a recombinant eukaryotic promoter in the nucleus and transported to the cytoplasm, where the viral replicase takes over in the same way as during normal replication of alphavirus RNA molecules. The levels of reporter gene expression is 10-200-fold higher in mouse muscle cells for alphavirus DNA vectors than with conventional DNA vectors (Johanning, et al., infra, Herweijer, et al., infra; Dubensky, T. W., Driver, D. A., Polo, J. M., Belli, B. A., Latham, E. M., Ibanez, C. E., Chada S., Brumm D., Banks T. A., Mento S. J., et al., Sinbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer,
J. Virol.
1996, 70: 508-519). Expression is transient in nature as seen in mouse quadriceps, where no expression is detected 16 days post-injection. Therefore, alphaviruses can be efficiently used as tools in gene therapy for safe short-term gene expression.
The present invention relates to RNA virus expression and particularly to an alphavirus expression system in which one can control viral RNA replication so that it is not lethal or not substantially lethal to the host cell, and is therefore amenable to large scale production of RNA and protein. However, any viral expression system which employs RNA self-replication as a mechanism for viral amplification and expression can be used according to the present invention, including alphaviruses (animal viruses) such as togaviruses, i.e. Sindbis, SFV, Eastern Equine Encephalitis Virus (EEEV) and Venezuelan Equine Encephalitis Virus (VEEV), and flaviviruses, i.e. yellow fever virus, tick borne encephalitis virus; as well as plant viruses such as tobamoviruses (tobacco mosaic virus family) and bromoviruses (brome mosaic virus family) and variants, derivatives or modifications thereof. Viral RNA replication can be regulated by the methods discussed below.
Therefore, it is one object of the present invention to provide a method for controlling replication of viral RNA wherein cells are engineered to contain nucleic acid molecules (for example incorporated into the genome or into one or more vectors within the cells) having one or more of the virus (e.g. alphavirus) non-structural protein genes under the control of an inducible promoter. An expression construct (which may be RNA or
Ciccarone Valentina C.
Jessee Joel A.
Hutson Richard G.
Invitrogen Corporation
Prouty Rebecca E.
Sterne Kessler Goldstein & Fox P.L.L.C.
LandOfFree
Regulated expression of recombinant proteins using RNA viruses does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Regulated expression of recombinant proteins using RNA viruses, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Regulated expression of recombinant proteins using RNA viruses will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2879248