Replication-competent adenoviral vectors
Inventors
Peng, Bo • Voltan, Rebecca • Ensoli, Barbara • Robert-Guroff, Marjorie
Assignees
Istituto Superiore di Sanita ISS • US Department of Health and Human Services
Publication Number
US-9732359-B2
Publication Date
2017-08-15
Expiration Date
2025-11-17
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Abstract
This invention provides improved replication-competent adenoviral vectors. The improved vectors have both a hybrid regulatory unit that provides for high level transgene expression. The vectors can be use, e.g., for therapeutic or prophylactic purposes.
Core Innovation
This invention provides improved replication-competent adenoviral vectors comprising a hybrid regulatory unit that includes a human cytomegalovirus (CMV) immediate early promoter and an adenovirus tripartite leader (tpl) sequence, which results in greatly enhanced gene expression. The hybrid regulatory unit controls expression of a transgene and is operably linked to it, enabling high levels of gene expression in replication-competent adenoviruses including types 2, 4, 5, and 7. The vectors may lack non-essential sequences such as the E3 region, into which the transgene can be inserted.
The problem addressed is that prior art replication-competent adenovirus vectors either used native or heterologous promoters but often failed to provide high-level transgene expression, especially for proteins like hepatitis B virus surface antigen. There was no suggestion or teaching in the prior art that incorporating a heterologous promoter, such as the CMV promoter combined with the adenovirus tpl sequence, in replication-competent adenoviral vectors would yield beneficial effects. The present invention solves this problem by combining the CMV immediate early promoter with adenovirus tripartite leader sequences to achieve significantly higher transgene expression compared to previous vectors.
The invention further addresses specific needs in the development of HIV vaccines by providing replication-competent adenoviral vectors expressing HIV genes, including regulatory genes such as nef and tat, as well as structural genes such as gag and env. The vectors enable high-level expression of these HIV genes, which are important for eliciting broad immune responses. Notably, embodiments include nef proteins that are not myristoylated to maintain epitopes while abrogating negative modulatory functions, and tat sequences that lack transactivation function. Thus, the invention combines enhanced expression capabilities with immunological considerations for vaccine development.
Claims Coverage
The patent comprises one independent claim reciting a method involving a replication-competent adenovirus vector with a hybrid regulatory cassette for transgene expression. The following main inventive features are claimed.
Replication-competent adenovirus vector with hybrid expression cassette
A replication-competent adenovirus vector that includes an endogenous adenovirus tripartite leader sequence and a hybrid expression cassette comprising a CMV immediate early promoter, a second adenovirus tripartite leader sequence, and a transgene that is expressed in a host cell.
Vector serotype selection
The adenovirus vector can be selected among adenovirus types 2, 4, 5, or 7.
Deletion of E3 region and insertion of transgene
The adenovirus vector lacks a functional E3 region, often deleted, where the transgene is inserted.
Expression of HIV genes as transgenes
The transgene can be an HIV gene, including regulatory genes such as nef or tat, with nef optionally not myristoylated and tat optionally lacking transactivation function, or structural genes such as gag or env.
Spliced tripartite leader sequence use
The second adenovirus tripartite leader sequence of the hybrid expression cassette is a spliced tripartite leader sequence, exemplified by the sequences of SEQ ID NO:1 or SEQ ID NO:2.
The claims cover a method employing replication-competent adenovirus vectors comprising a hybrid regulatory unit containing CMV promoter and adenoviral tripartite leader sequences, with optional deletion of the E3 region for transgene insertion, targeting a range of adenovirus serotypes, and expressing HIV genes including modified nef and tat, thereby enhancing transgene expression in host cells.
Stated Advantages
The vectors provide high-level transgene expression relative to prior replication-competent adenovirus vectors lacking the hybrid regulatory unit.
Replication-competent vectors achieve higher gene expression in vivo with lower adenovirus doses compared to replication-defective vectors, reducing adverse immunopathology.
In vivo adenovirus vector replication stimulates production of pro-inflammatory cytokines that augment immune responses.
The hybrid regulatory unit's combined CMV promoter and adenoviral major late promoter enable increased tpl-containing mRNA templates each replication cycle for efficient transgene expression.
The vectors enable high-level expression of HIV genes, facilitating improved vaccine immunogenicity including induction of cellular and humoral immune responses.
Documented Applications
Use as vectors for therapeutic or prophylactic purposes, such as vaccine development.
Use for expression of HIV genes, including regulatory genes (nef and tat) and structural genes (gag and env), to induce immune responses against HIV.
Use for eliciting cellular immune responses, including enhancement of co-administered antigens' immunogenicity, demonstrated in animal models including mice, rhesus macaques, cynomolgus monkeys, and chimpanzees.
Use in vaccine strategies to prime immune responses for HIV infections, including incorporation of modified Nef constructs to enhance immune response quality.
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