Adenoviral expression vector and methods and cell lines for production

Inventors

Exline, Colin • Liu, Huizhu • Stevens, Sean

Assignees

Lung Biotechnology PBC

Abstract

An adenovirus expression vector is provided. The adenovirus expression vector may include: a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding a protein or an RNA is provided. A method of synthesizing an adenovirus vector is also provided. The synthesis may include: a) producing a plurality of overlapping adenovirus sub-fragments, each sub-fragment comprising a portion of the full genome of the adenovirus; b) circularizing the sub-fragments to form plasmid structures; and c) assembling the circularized sub-fragments into a linear structure, wherein the vector comprises a combination of two or more sub-fragments. A mammalian cell line configured to replicate adenoviral vectors, wherein the cell line comprises nucleotide sequences expressing E1A and E1B gene products but is devoid of other adenovirus sequences is also provided.

Core Innovation

The disclosure describes a non-replicating adenovirus expression vector comprising one or more mutations that render the adenovirus replication incompetent, and at least one nucleotide sequence encoding a gene product. The vector includes a nucleotide sequence having at least 95% sequence identity over the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or the entire complementary sequence thereto, and replication-incompetent mutations include deletion of the E1 gene and/or deletion of the E3 gene.

The gene product is expressed from at least one transgene or RNA expression cassette within the replication-incompetent adenovirus vector. The expression cassette may include a CAG promoter/enhancer, SV40 promoter region, and polyA signals, and the nucleotide sequence encoding the gene product may be inserted to replace E1 while leaving pIX intact.

The disclosure further provides an adenoviral vector synthesis strategy using overlapping adenovirus sub-fragments that are circularized into plasmid structures and assembled into a linear genome for high-efficiency bacterial DNA replication. For mammalian production, the disclosure describes a producer cell line strategy that expresses E1A and E1B but lacks other adenoviral sequences, implemented via a lentiviral system in cells such as HeLa, to reduce recombination contamination restoring replication competence.

Claims Coverage

The independent claim defines a non-replicating adenovirus expression vector with replication-incompetent adenovirus mutations and a gene product encoding sequence, constrained by at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 (or the complementary sequence). Six inventive features are identified across the claims, with dependent claims refining sequence identity, adenovirus serotype, replication-incompetent mutations, expression-cassette content, and insertion placement.

Overall, claim coverage centers on a non-replicating adenovirus expression vector defined by replication-incompetent adenovirus mutations and whole-sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 (or complements), with refinements to adenovirus serotype 5, E1/E3 deletions, polyadenylation signal inclusion, and insertion placement that replaces E1 while leaving pIX intact.

Stated Advantages

Documented Applications