Capsid-free AAV vectors, compositions, and methods for vector production and gene delivery
Inventors
Garcia, Luis • Beley, Cyriaque • Voit, Thomas • Kotin, Robert Michael • Li, Lina
Assignees
Centre National de la Recherche Scientifique CNRS • Universite Pierre et Marie Curie • Institut National de la Sante et de la Recherche Medicale INSERM • Association Institut de Myologie • National Institutes of Health NIH • US Department of Health and Human Services
Publication Number
US-9598703-B2
Publication Date
2017-03-21
Expiration Date
2032-03-12
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Abstract
An isolated linear nucleic acid molecule comprising in this order: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest and a second AAV ITR, wherein said nucleic acid molecule is devoid of AAV capsid protein coding sequences. The said nucleic acid molecule can be applied to a host at repetition without eliciting an immune response. Methods for producing and purifying this nucleic acid molecule, and use of the same for therapeutic purposes are also provided.
Core Innovation
The invention relates to an isolated linear nucleic acid molecule comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest, and a second AAV ITR, wherein the nucleic acid molecule is devoid of AAV capsid protein coding sequences. These capsid-free AAV vectors, denoted as rAAV0, enable the delivery of exogenous DNA sequences for in vitro, ex vivo, or in vivo applications such as therapeutic gene delivery. The rAAV0 vectors are distinct from conventional plasmids and AAV vectors as they lack prokaryotic DNA, AAV capsid and rep genes, and comprise self-contained elements necessary for replication and expression, including ITRs that form hairpins and confer resistance to nucleases.
The problem solved arises from limitations of conventional AAV vectors, which include eliciting an immune response primarily due to capsid proteins, limited packaging capacity (approximately 4.5 kb), inhomogeneous vector preparations, and slow gene expression requiring conversion from single-stranded to double-stranded DNA. Additionally, pre-existing immunity to AAV serotypes and the restricted ability to re-administer vectors without immunosuppression limit therapeutic efficacy. The invention overcomes these drawbacks by dispensing with the capsid, thereby increasing packaging capacity, reducing immune response, and enabling repeated administration of vectors without the need for transduction aids or immunosuppressants.
The invention further discloses methods for producing and purifying these capsid-free AAV vectors from host cells, such as Sf9 insect cells, which are engineered to contain the rAAV0 genome but lack capsid protein expression, and provide Rep proteins to facilitate rescue and amplification of the rAAV0 vector. Purification can be carried out by isolating the linear DNA vectors or as exosomes or microparticles that carry the vectors. The rAAV0 vector exhibits efficient cell transduction without requiring the viral capsid, and can be administered via various routes, including intramuscular, intra-arterial, intrathecal, and systemic delivery, with well-characterized homogeneity and absence of immunogenicity.
Claims Coverage
The patent contains multiple independent claims covering the isolated nucleic acid molecule, methods of production, and methods of use, together defining capsid-free AAV vectors devoid of capsid protein coding sequences and their application.
Isolated linear capsid-free nucleic acid molecule structure
An isolated linear nucleic acid molecule comprising a first AAV inverted terminal repeat (ITR), a nucleotide sequence of interest or expression cassette, and a second AAV ITR, wherein the nucleic acid molecule is devoid of AAV capsid protein coding sequences and optionally rep sequences, existing as single-stranded or double-stranded linear DNA not encapsidated in a viral capsid.
Method of producing capsid-free AAV vectors
A method comprising providing cells containing two AAV ITRs flanking an expression cassette devoid of capsid protein coding sequences without expression of capsid proteins in the host cells, optionally providing Rep genes or proteins, culturing cells under conditions permitting replication and release of the linear capsid-free AAV vector DNA, and collecting the rescued vector.
Method of mediating expression of exogenous DNA in mammalian cells and subjects
Delivering an effective amount of a capsid-free AAV vector comprising two AAV ITRs and an exogenous DNA cassette without capsid protein encoding sequences to cells or tissues, under conditions permitting expression without eliciting an immune response, including delivery without transfection aids or physical facilitation.
The claims collectively encompass the isolated capsid-free AAV linear nucleic acid molecules with flanking ITRs, their methods of production involving Rep protein complementation but absence of capsid proteins, and methods for delivering these vectors to cells or subjects resulting in expression of exogenous DNA without triggering capsid-specific immune responses and overcoming packaging size limitations associated with conventional AAV vectors.
Stated Advantages
rAAV0 vectors do not elicit a host immune response triggered by viral capsid proteins, enabling repeated administrations without immunosuppression.
They allow incorporation of nucleotide sequences exceeding conventional AAV packaging capacity, accommodating from about 4.2 kb up to about 15 kb or more, overcoming size limitations.
Productions yield substantially homogeneous vector preparations that can be fully characterized by standard DNA analytical methods.
rAAV0 vectors transduce cells efficiently without need for transduction aids or viral capsid mediation, and reach the nucleus effectively.
Use of eukaryotic-origin DNA without bacterial sequences reduces immunogenicity compared to plasmid DNA.
Documented Applications
In vitro, ex vivo, and in vivo delivery of therapeutic or experimental nucleic acid sequences for protein or RNA expression.
Gene therapy applications including treatment of genetic and acquired muscle diseases such as Duchenne muscular dystrophy via delivery of genes like dystrophin or U7-based exon-skipping sequences.
Delivery of therapeutic nucleic acids to neural cells for treatment of neurological and neurodegenerative diseases including Parkinson's, Huntington's, Alzheimer's, ALS, epilepsy, stroke, and others.
Vaccination by intramuscular administration of rAAV0 vectors expressing antigens without eliciting immune response against the vector.
Modification of stem cells ex vivo or in vivo for gene correction, including delivery of DNA-cutting enzymes like meganucleases, zinc finger nucleases, or TAL effectors.
Delivery of rAAV0 via exosomes or microparticles targeted to specific tissues or cells.
Therapeutic applications in diseases beyond muscular dystrophies such as Pompe disease, cystic fibrosis, solid tumors, and immune tolerance induction.
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