Alphaherpesvirus LAP Promoters for Long-Term AAV Transgene Expression
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Solution Overview
Problem
Existing gene delivery vectors, particularly adeno-associated virus (AAV) vectors, face limitations in payload size and are prone to repression of transcription, making them unsuitable for long-term, chronic expression of therapeutic transgenes.
Innovation Solution
Utilization of latency-associated promoter (LAP) sequences from alphaherpesvirus genomes, such as PRV, which are smaller in size and less prone to transcriptional repression, integrated into AAV vectors to drive long-term expression of target genes in various cell types.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If traditional promoters (e.g., EF1α) are used in AAV vectors, then strong initial transcription is achieved, but transcriptional repression occurs over time preventing long-term expression
Solution Approach 1:
The patent changes the biochemical parameters of the promoter system by replacing traditional mammalian promoters with alphaherpesvirus LAP sequences. This substitution fundamentally alters the transcriptional regulation mechanism, enabling sustained activity without repression. The LAP sequences maintain high transcriptional output over extended periods (months to years) unlike traditional promoters that undergo silencing, thus resolving the contradiction between duration and reliability of expression.
2Adaptability or versatility
If AAV vector payload is increased to include larger promoter sequences, then more comprehensive transcriptional control is achieved, but vector capacity limitations are exceeded
Solution Approach 1:
The patent extracts only the essential functional elements needed for long-term transcriptional activity. By identifying and utilizing the core LAP sequences from alphaherpesvirus genomes, the invention achieves comprehensive transcriptional control with minimal payload. These extracted promoter elements provide sustained expression capability without requiring the full complexity of traditional promoter systems, thus fitting within AAV capacity constraints while maintaining adaptability.
3Duration of action of moving object
If alphaherpesvirus LAP sequences are used, then long-term transcriptional expression is achieved, but potential viral integration and safety concerns arise
Solution Approach 1:
The patent employs a disposable, non-integrating approach by using self-contained AAV vectors with alphaherpesvirus promoter sequences that do not require or enable viral integration. The LAP-driven expression system functions autonomously within the vector, providing long-term expression (months to years) without the safety concerns associated with integrative viral mechanisms. This resolves the contradiction by achieving durable expression through non-integrative, controlled vector systems.
Data Source
AI summary
Provided are recombinant nucleic acids (e.g., vectors), and related methods, for expression of a target gene in a host cell. The recombinant nucleic acids comprise a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for the expression of the target gene in the host cell.