Alphavax, Incorporated

Other

Sloan-kettering Institute for Cancer Research

Maureen Maughan

Jonathan F Smith

Kurt Iver Kamrud

Sergey A Dryga

Robert E Johnston

Nancy L Davis

Jon O Rayner

Ronald I Swanstrom

Robert A Olmsted

Ian Caley

Paula M Keith

Harold Alterson

Kim Butler

Francesca Avogadri

The present invention provides an isolated RNA molecule comprising: a) an alphavirus 5' replication recognition sequence, wherein at least one initiation codon has been removed from the 5&#x2032; replication recognition sequence; b) a nucleotide sequence encoding an alphavirus structural protein; and c) an alphavirus 3&#x2032; replication recognition sequence, with the proviso that the RNA molecule does not contain a promoter that directs transcription of the nucleotide sequence of (b), and wherein the alphavirus 5&#x2032; and 3&#x2032; replication recognition sequences of (a) and (c) direct replication of the RNA molecule in the presence of alphavirus non-structural proteins.

Promoterless cassettes for expression of alpha virus structural proteins

The present invention provides an isolated RNA molecule comprising: a) an alphavirus 5' replication recognition sequence, wherein at least one initiation codon has been removed from the 5&#x2032; replication recognition sequence; b) a nucleotide sequence encoding an alphavirus structural protein; and c) an alphavirus 3&#x2032; replication recognition sequence, with the proviso that the RNA molecule does not contain a promoter that directs transcription of the nucleotide sequence of (b), and wherein the alphavirus 5&#x2032; and 3&#x2032; replication recognition sequences of (a) and (c) direct replication of the RNA molecule in the presence of alphavirus nonstructural proteins.

Promoterless cassettes for expression of alphavirus structural proteins

Provided herein are methods for producing alphavirus replicon particles in high yield; replicon RNAs are electroporated into permissive cells, where the cells are at a relatively high density, together with at least one helper nucleic acid providing the necessary functions for packaging. After a growth period in appropriate medium, alphavirus replicon particles are harvested from the surfaces of the cells in which they were produced using a salt wash in which the salt concentration is from about 0.2 to about 5 M sodium chloride, calcium chloride, magnesium chloride, potassium chloride, ammonium acetate, ammonium bicarbonate, among others. After dilution, if necessary, the particles can be purified by a suitable chromatographic technique.

Alphavirus particles and methods for preparation

The present invention provides methods and compositions comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a g&agr;g gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the g&agr;g gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the g&agr;g gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit protease, integrase, RNase H and/or reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.

Alphavirus vectors and virosomes with modified HIV genes for use in vaccines

Growing community of inventors

Durham, NC, United States of America

Maureen Maughan