Engineered untranslated regions (UTR) for AAV production
Inventors
Maranga, Luis • Cecchini, Sylvain • Dismuke, David
Assignees
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Publication Number
US-12060567-B2
Publication Date
2024-08-13
Expiration Date
Abstract
The present disclosure describes methods and systems for use in the production of recombinant adeno-associated virus (rAAV) particles. In certain embodiments, the production process and system include engineered untranslated regions (UTR) which allow for the controlled expression of AAV capsid proteins, such as VP1, VP2 and VP3. In certain embodiments, the production process and system include engineered untranslated regions (UTR) which allow for the controlled expression of non-structural AAV replication proteins, such as Rep78 and Rep52.
Core Innovation
The invention provides expression control sequences comprising a start codon and an engineered 5′ untranslated-region polynucleotide encoding a hairpin structure. The hairpin comprises defined flanking, stem, and loop regions, positioned in relation to a start codon alongside promoter, leader, and Kozak features. These regulatory sequences are deployed in viral expression constructs to regulate adeno-associated virus protein production and modulate capsid-protein ratios.
The disclosure describes the regulation of adeno-associated virus protein expression, encompassing capsid-protein stoichiometry and relative expression of replication proteins. Comparisons of engineered hairpin leader variants demonstrate differing expression outcomes, including reduced or absent protein expression across specific configurations. Because the background section is omitted from the supplied content, prior deficiencies are not explicitly described in the patent.
Claims Coverage
The claims recite three inventive features across independent claims 1, 17, and 19, which cover expression control sequences containing an engineered 5′ UTR hairpin with distinct sequence definitions.
Hairpin with a specified loop region
Claim 1 covers an expression control sequence comprising a start codon and an engineered 5′ UTR polynucleotide encoding a hairpin with flanking, stem, and loop regions, wherein the loop region is defined by specified sequence alternatives or identity criteria.
Hairpin with a specified 3′ flanking region
Claim 17 covers an expression control sequence comprising a start codon and an engineered 5′ UTR polynucleotide encoding a hairpin with 5′ and 3′ flanking regions, a stem, and a loop, wherein the 3′ flanking region satisfies specified sequence criteria.
Hairpin selected from disclosed sequences
Claim 19 covers an expression control sequence comprising a start codon and an engineered 5′ UTR polynucleotide encoding a hairpin selected from a disclosed sequence set, with defined flanking, stem, and loop regions.
The three independent claims cover engineered 5′ UTR hairpin expression controls distinguished by their loop-region definition, 3′ flanking-region definition, or selection from the disclosed sequence set, with dependent claims extending coverage to promoter options, viral expression constructs encoding AAV proteins, and protein production methods.
Stated Advantages
The disclosed expression controls are intended to improve control of protein expression and rAAV production.
The disclosed constructs may improve AAV yields.
Documented Applications
Regulating AAV capsid-protein and replication-protein expression in viral expression constructs intended for rAAV production.
Protein production from viral expression constructs in viral production cells, including mammalian and insect cell systems such as a baculovirus expression system.
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