Closed-ended, linear, duplex adenoassociated virus DNA, and uses thereof
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-11549125-B2
Publication Date
2023-01-10
Expiration Date
2037-08-09
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Abstract
Closed-ended, linear, duplex (CELID) DNA molecules, recombinant AAV (rAAV), particles comprising CELID DNA, methods of making such molecules and particles, and therapeutic applications of such particles.
Core Innovation
This disclosure provides closed-ended, linear, duplex (CELID) DNA molecules, recombinant adeno-associated virus (rAAV) particles comprising CELID DNA, methods of producing such molecules and particles, and therapeutic applications thereof. The CELID DNA comprises heterologous DNA flanked by inverted terminal repeats (ITRs) that form T-shaped hairpin structures, containing AAV Rep protein binding sites and terminal resolution sites (trs). These CELID DNA molecules are produced by culturing mammalian or invertebrate cells expressing AAV Rep proteins with nucleic acid molecules comprising heterologous DNA flanked by ITRs, allowing replication and isolation of such DNA molecules. The CELID DNA is then used to produce rAAV particles by introducing it into cells that express AAV Rep and Cap proteins, resulting in rAAV particles that package CELID DNA rather than contaminating plasmid or bacterial DNA.
The problem being addressed is that conventional recombinant AAV production methods relying on plasmid transfection result in encapsidation of non-vector DNA such as plasmid backbone DNA, which may induce innate immune responses and reduce safety and efficacy of gene therapy vectors. Additionally, plasmid-derived DNA sequences exhibit prokaryotic methylation patterns that are undesirable in therapeutic products. Improved methods are needed to reduce or eliminate these contaminating sequences, thereby providing rAAV particles with more homogeneous and safer DNA cargo. The disclosed CELID DNA methods overcome these issues by producing AAV vector genomes of eukaryotic origin, thereby avoiding bacterial DNA contamination and associated impurities like endotoxin, and enhancing the purity and safety profile of rAAV particles.
Claims Coverage
There is one independent claim focusing on a recombinant AAV particle produced by a specific method involving CELID DNA and AAV capsid proteins. The claims detail inventive features relating to the composition of the rAAV particles, production methods, and DNA purity metrics.
Production of rAAV particles comprising CELID DNA
A method comprising culturing a cell with (i) closed ended, linear duplex (CELiD) DNA containing heterologous DNA, and (ii) one or more nucleic acid molecules encoding AAV capsid proteins, under conditions suitable for formation of AAV particles containing the CELID DNA.
rAAV particles with minimal contaminating DNA
Recombinant AAV particles comprising CELID-derived DNA including heterologous DNA, wherein DNA from sources other than AAV or the heterologous DNA constitutes less than 10% (and optionally less than 1%, 0.1%, or 0.01%) of total DNA in the particle.
Use of stably inserted capsid protein genes in producer cells
Producer cells used in the method may have nucleic acid molecules encoding one or more AAV capsid proteins stably inserted into their genome.
Involvement of Rep protein expression in production
Cells may comprise one or more polynucleotide molecules encoding AAV Rep proteins, and culture conditions allow replication of CELID DNA and expression of Rep and Cap proteins.
CELID DNA encoding therapeutic proteins or RNAs
The heterologous DNA packaged in rAAV may encode proteins or therapeutic RNAs including siRNA, RNAi, shRNA, miRNA, aptamers, or ribozymes.
Use of various mammalian or invertebrate cells
The cells used for producing rAAV particles may be mammalian or invertebrate cells such as insect cells.
The claims collectively cover methods of producing rAAV particles comprising CELID DNA with high purity and minimal contaminating DNA, the compositions of such rAAV particles, use of producer cells genetically modified for capsid and Rep protein expression, and therapeutic compositions comprising nucleic acids encoding proteins or RNAs within CELID DNA.
Stated Advantages
Prevention of encapsidation of non-vector DNA, including plasmid backbone DNA, improving the purity of recombinant AAV particles.
Reduction or elimination of prokaryotic DNA methylation patterns and bacterial impurities such as endotoxins in the final gene therapy product.
Production of exonuclease-resistant, closed-ended, linear duplex DNA molecules that serve as a stable vector genome for rAAV particles.
Improved safety of recombinant AAV particles due to reduced contaminating DNA, lowering risk of innate immune activation.
Ability to produce CELID DNA and rAAV particles in mammalian or invertebrate cell lines, providing flexibility in manufacturing.
Documented Applications
Therapeutic delivery of nucleic acids to individuals by administering rAAV particles containing CELID DNA encoding therapeutic molecules to treat or ameliorate disease.
Eliciting immune responses in individuals via administration of rAAV particles comprising CELID DNA encoding immunogenic proteins for vaccination purposes.
In vitro, ex vivo, or in vivo delivery of exogenous DNA sequences to cells, tissues, organs, or individuals using recombinant AAV particles produced with CELID DNA.
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