Flavivirus expression and delivery system

Abstract

The present invention provides a gene expression system comprising: a) a self-replicating expression vector of flavivirus origin which includes the flavivirus 5′ untranslated region (UTR), at least a portion of the 5′ coding region for flavivirus core protein, the nucleotide sequence coding for the flavivirus non-structural proteins, and the complete or most of the 3′-terminal sequence of the flavivirus 3′ UTR, required for self-replication of flavivirus genomic material, which vector is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities; and b) at least a second vector that is capable of expressing flavivirus structural protein(s) and any other proteins required for packaging of the self-replicating expression vector into flavivirus viral particles which vector is engineered to prevent recombination with the self-replicating vector when in its presence.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation of U.S. Ser. No. 09 / 580,476, filed May 26, 2000, which is a continuation of International Application No. PCT / AU98 / 00993 (published as International Publication No. WO 99 / 28487), filed 30 Nov. 1998 and designating the United States, which in turn claims priority from Australian Application Nos. PP 0627, filed 28 Nov. 1997 and PP 6096, filed 23 Sep. 1998, the teachings of all of which are incorporated herein by reference.[0002] The present invention generally relates to the field of gene expression and in particular to Flavivirus gene expression and delivery systems and to virus like particles produced from such systems. [0003] The present invention generally relates to the field of gene expression and in particular to Flavivirus gene expression and delivery systems and to virus like particles produced from such systems. [0004] Improved methodologies for maximising recombinant gene expression are...

AI Technical Summary

Benefits of technology

[0043] In use, the replicon is introduced into a host cell where gene expression and hence protein production take place. Because the vector is capable of self-replication, multiple copies of the replicon will also be generated. This leads to an exponential increase in the number of replicons in the host cell as well as an exponential increase in the amount of protein that is produced.

[0052] Optimal flavivirus replicon design for transfection into eukaryotic cells might also include sequences inserted into the replicon such as: sequences to promote expression of the heterologous gene of interest, including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence; internal ribosomal entry site (IRES) of picornaviruses; an alphavirus subgenomic 26S promoter to enhance expression of inserted genes if cotransfection with alphavirus replicon RNA is used.

[0059] The replicon described herein may also be engineered to express multiple nucleotide sequences allowing co-expression of several proteins such as a plurality of antigens together with cytokines or other immunomodulators to enhance the generation of an immune response. Such a replicon might be particularly useful for example in the production of various proteins at the same time or in gene therapy applications.

[0061] The nucleotide sequence may also code for one or more amino acid sequences that serve to enhance the effect of the protein being expressed. For example, ubiquitination of viral proteins expressed from DNA vectors results in enhancement of cytotoxic T-lymphocyte induction and antiviral protection after immunization. Thus, in a preferred embodiment of the invention the replicon may encode ubiquitin in association with the protein to be expressed thus targeting the resulting fusion protein to proteosomes for efficient processing and uptake by the MHC class I complexes.

[0064] To optimise expression of the flavivirus structural genes, the second vector might include such sequences as: sequences to promote expression of the genes of interest, including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence. Preferably, the second vector contains separate regulatory elements associated with each of the different structural genes expressed by the vector. Most preferably, the flavivirus C gene and the prME genes are placed under the control of separate regulatory elements in the vector.

[0071] The replicon containing flavivirus like particles that contain nucleotide coding sequence for immunogenic polypeptide(s) as active ingredients may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The flavivirus replicon therapeutic(s) may also be mixed with excipients that are pharmaceutically acceptable and compatible with the replicon encapsulated viral particle. Suitable excipients are, for example, water, saline, dextrose glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the therapeutic may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvant which enhance the effectiveness of the therapeutic.

Problems solved by technology

While procaryotic and yeast expression systems are extremely efficient and easy to use, these systems suffer from a number of disadvantages, including an inability to glycosylate proteins, inefficient cleavage of “pre” or “prepro” sequences from proteins (eg., inefficient post translational modification), and a general inability to secrete proteins.

This system is arguably one of the most efficient in protein production, but is limited only to use in insect cell lines.

Unfortunately, insect cell lines glycosylate proteins differently from mammalian cell lines thus this system has not proven useful for the production of many mammalian proteins.

Another disadvantage of this system is that it relies on the use of homologous recombination for the construction of recombinant virus stocks.

Thus, this system often proves very laborious when large numbers of genetic variants have to be analysed.

The main problem with this system, however, is that it uses recombinant viruses that express the heterologous gene upon infection.

The main problem with these expression systems is that the viruses used in the expression system are cytopathic and often compete out the host protein synthesis.

Another major disadvantage of these systems includes possible contamination with infectious particles containing packaged full-length genomic RNA (in other words, infectious virus) due to the high probability of recombination between replicon and helper RNAs.

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