Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-08
2002-10-15
Wortman, Donna C. (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100
Reexamination Certificate
active
06465634
ABSTRACT:
TABLE OF CONTENTS
A. Sources of Wild-Type Alphavirus
B. Selection of Alphaviruses with a Desired Phenotype
1. Biological Selection of Virus Variants
a. Selection from Virus Stocks Containing DI Particles
b. Selection from Virus Stocks Not Containing DI Particles
2. Genetic Selection of Virus Variants
3. Genetic Selection of Variants Using Virus-Derived Vectors
4. Use of Viral Variants
C. Alphavirus Vector Constructs and Alphavirus RNA Vector Replicons
1. 5′ Promoters Which Initiate Synthesis of Viral RNA
2. Sequences Which Initiate Transcription
3. Alphavirus Nonstructural Proteins
a. nsP1
b. nsP2
c. nsP3
d. nsP4
4. Viral Junction Regions
5. Alphavirus RNA Polymerase Recognition Sequence, and Poly(A) Tract
D. Eukaryotic Layered Vector Initiation Systems
E. Recombinant Alphavirus Particles, and Generation and Use of ‘Empty’ Togavirus Particles or Togaviruses Particles Containing Non-Homologous Viral RNA
F. Heterologous Sequences
1. Lymphokines
2. Toxins
3. Prodrug Converting Enzymes
4. Antisense Sequences
5. Ribozymes
6. Proteins and Other Cellular Constituents
a. Altered Cellular Components
b. Antigens from Foreign Organisms or Other Pathogens
7. Sources for Heterologous Sequences
G. Alphavirus Packaging/Producer Cell Lines
H. Pharmaceutical Compositions
I. Methods for Utilizing Gene Delivery Vehicles
1. Immunostimulation
2. Blocking Agents
3. Expression of Palliatives
a. Inhibitor Palliatives
b. Conditional Toxic Palliatives
4. Expression of Markers
5. Immune Down-Regulation
6. Replacement or Augmentation Gene Therapy
7. Lymphokines and Lymphokine Receptors
8. Suicide Vectors
9. Gene Delivery Vehicles to Prevent the Spread of Metastatic Tumors
10. Administration of Gene Delivery Vehicles
11. Modulation of Transcription Factor Activity
12. Production of Recombinant Proteins
J. Deposit Information
EXAMPLES
Example 1
Isolation and Characterization of SIN-1
A. Isolation, Plaque Purification, and Characterization of SIN-1 from a Wild-Type Sindbis Virus Stock
B. Molecular Cloning of SIN-1
C. Sequence of the SIN-1 Phenotype
D. Characterization and Genetic Mapping of the SIN-1 Phenotype with Molecular Clones
Example 2
Isolation and Characterization of Positive Strand RNA Viruses Which Exhibit Reduced Inhibition of Host Macromolecular Synthesis
A. Biological Selection of Virus Variants
B. Genetic Selection of Virus Variants
C. Genetic Selection of Variants Using Virus-Derived Vectors
1 Vectors Expressing an Immunogenic Protein
2. Vectors Expressing a Selectable Marker
Example 3
Preparation of SIN1-Based RNA Vector Replicons
A. Construction of the SIN-1 Basic Vector
Example 4
Preparation of SIN1-Based DNA Vectors
A. Construction of Plasmid DNA SIN-1 Derived Expression Vectors
B. Expression of Heterologous Proteins in Cells Transfected with PBG/SIN-1 ELVS 1.5-SEAP, PBG/SIN-1 ELVS 1.5-LUC OR PBG/SIN-1 ELVS 1.5-B-Gal Expression Vectors
Example 5
Modifications of Plasmid DNA SIN-1Derived Expression Vectors
Example 6
Construction of Alphavirus Packaging Cell Lines
A. Construction of Vector-Inducible Alphavirus PCL
B. Construction of PCL with Operably-Linked Selection Marker
C. Construction of “Split Structural Gene” PCL Configurations
D. Construction of PCL with “Hybrid” Structural Proteins
E. Production of Packaged Alphavirus Vectors from PCL
Example 7
Construction of Alphavirus Producer Cell Lines
A. Alphavirus DNA Vectors with Single Level Regulation
B. Alphavirus DNA Vectors with Two Level Regulation
Example 8
Methods for the Generation of Alphavirus Derived Empty or Chimeric Viral Particles
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to recombinant DNA technology; and more specifically, to the development of recombinant vectors useful for directing the expression of one or more heterologous gene products.
BACKGROUND OF THE INVENTION
Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. These viruses are distributed worldwide, and persist in nature through a mosquito to vertebrate cycle. Birds, rodents, horses, primates, and humans are among the defined alphavirus vertebrate reservoir/hosts.
Twenty-six known viruses and virus subtypes have been classified within the alphavirus genus utilizing the hemagglutination inhibition (HI) assay. This assay segregates the 26 alphaviruses into three major complexes: the Venezuelan equine encephalitis (VEE) complex, the Semliki Forest (SF) complex, and the western equine encephalitis (WEE) complex. In addition, four other viruses, eastern equine encephalitis (EEE), Barmah Forest, Middelburg, and Ndumu, receive individual classification based on the HI serological assay.
Members of the alphavirus genus also are classified based on their relative clinical features in humans: alphaviruses associated primarily with encephalitis, and alphaviruses associated primarily with fever, rash, and polyarthritis. Included in the former group are the VEE and WEE complexes, and EEE. In general, infection with this group can result in permanent sequelae, including behavior changes and learning disabilities, or death. In the latter group is the SF complex, comprised of the individual alphaviruses Semliki Forests Sindbis, Ross River, Chikungunya, O'nyong-nyong, and Mayaro. With respect to this group, although serious epidemics have been reported, infection is in general self-limiting, without permanent sequelae.
Sindbis virus is the prototype member of the Alphavirus genus of the Togaviridae family. Its replication strategy after infection of cells (see
FIG. 1
) has been well characterized in chicken embryo fibroblasts (CEF) and baby hamster kidney (BHK) cells, where Sindbis virus grows rapidly and to high titer, and serves as a model for other alphaviruses. Briefly, the genome from Sindbis virus (like other alphaviruses) is an approximately 12 kb single-stranded positive-sense RNA molecule which is capped and polyadenylated, and contained within a virus-encoded capsid protein shell. The nucleocapsid is further surrounded by a host-derived lipid envelope into which two viral-specific glycoproteins, E1 and E2, are inserted and anchored to the nucleocapsid. Certain alphaviruses (e.g., SF) also maintain an additional protein, E3, which is a cleavage product of the E2 precursor protein, PE2. After virus particle absorption to target cells, penetration, and uncoating of the nucleocapsid to release viral genomic RNA into the cytoplasm, the replicative process is initiated by translation of the nonstructural proteins (nsPs) from the 5′ two-thirds of the viral genome. The four nsPs (nsP1-nsP4) are translated directly from the genomic RNA template as one of two polyproteins (nsP123 or nsP1234), and processed post-translationally into monomeric units by an active protease in the C-terminal domain nsP2. A leaky opal (UGA) codon present between nsP3 and nsP4 of most alphaviruses accounts for a 10 to 20% abundance of the nsP1234 polyprotein, as compared to the nsP123 polyprotein. Both of the nonstructural polyproteins and their derived monomeric units may participate in the RNA replicative process, which involves binding to the conserved nucleotide sequence elements (CSEs) present at the 5′ and 3′ ends, and a junction region subgenomic promoter located internally in the genome (discussed further below).
The positive strand genomic RNA serves as template for the nsP-catalyzed synthesis of a full-length complementary negative strand. Synthesis of the complementary negative strand is catalyzed after binding of the nsP complex to the 3′ terminal CSE of the positive strand genomic RNA. The negative strand, in turn, serves as template for the synthesis of additional positive strand genomic RNA and an abundantly expressed 26S subgenomic RNA, initiated internally at the junction region promoter. Synthesis of additional positive strand genomic RNA occurs after binding of the nsP complex to the 3′ terminal CSE of the complementary negative strand genomic RNA template. Synthesis of the subgenomic mRNA from the negative strand genomic RNA temp
Belli Barbara A.
Dryga Sergey A.
Dubensky, Jr. Thomas W.
Frolov Ilya
Polo John M.
Blackburn Robert P.
Chiron Corporation
Dollard Anne S.
Pasternak Dahna
Wortman Donna C.
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