Nucleic acid molecule comprising asymmetrically modified ITR for improving expression rate of inserted gene, and use thereof
Inventors
Bae, Suk Chul • Lee, You Soub • Chi, Xinzi • Yoo, Seo Yeong • Kim, Woo-Jin
Assignees
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Publication Number
US-11993783-B1
Publication Date
2024-05-28
Expiration Date
Abstract
Described herein is a nucleic acid molecule including an asymmetrically modified inverted terminal repeat (ITR). An AAV vector including the nucleic acid molecule has advantages of increased productivity and expression efficiency of a transgene, and decreased genotoxicity, by having an asymmetric ITR in which any one of two ITRs is modified. Also, described herein is are compositions and vectors.
Core Innovation
The disclosed invention relates to a nucleic acid molecule in which a gene expression cassette is positioned between a first inverted terminal repeat (ITR) and a second ITR of an adeno-associated virus (AAV). The gene expression cassette contains a heterologous polynucleotide sequence, and the first ITR is an AAV wild-type TTR while the second ITR has a nucleotide sequence, or a complementary sequence, with at least about 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs: 1 to 9.
The second ITR is configured to include a terminal resolution site (trs) sequence and a rep-binding element (RBE) sequence while all of the B, B′, C, C′, and RBE′ regions are deleted. Either the A or the A′ region is deleted, with the deletion selected based on whether it is closer to the 3′-end of the (+) strand of the transgene or the 5′-end of the (−) strand of the transgene, and the second ITR exists as an open-end without forming any of the stem region and the loop region of the stem-loop structure while retaining functional activities of the trs and the RBE.
The background problem addressed is that AAV vectors can be affected by genotoxicity and by concatemer formation associated with long-term persistence, and that AAV productivity and transgene expression efficiency can be improved. The disclosed asymmetrically modified ITR design suppresses concatemer formation and related genotoxicity and integration-associated signals while increasing AAV productivity and transgene expression efficiency, including a reduction in persistence across passages as described for the provided comparisons.
Claims Coverage
The provided independent claim defines a tailored AAV ITR architecture with asymmetrically deleted regions, constrained sequence identity to SEQ ID NOs 1 to 9, retention of trs and RBE function, and open-end structural requirements for the second ITR. Dependent claims refine this architecture by adding stricter identity options, specifying AAV serotype sources, and defining elements of the gene expression cassette and payload, including vector and composition scopes.
Asymmetrically modified open-end second ITR retaining trs and RBE
A nucleic acid molecule with a gene expression cassette between a first ITR and a second ITR, where the second ITR consists essentially of a nucleotide sequence, or complementary sequence, having at least about 90% sequence identity to SEQ ID NOs: 1 to 9, the second ITR comprises a terminal resolution site (trs) sequence and a rep-binding element (RBE) sequence, all B, B′, C, C′, and RBE′ regions are deleted, and either A or A′, whichever is closer to the 3′-end of the (+) strand of the transgene or the 5′-end of the (−) strand, is deleted; the second ITR exists as an open-end without forming any of the stem region and the loop region of the stem-loop structure while retaining functional activities of the trs and the RBE.
Second ITR with 100% identity to SEQ ID NOs
The second ITR nucleotide sequence, or complementary sequence, has 100% identity to any of SEQ ID NOs: 1 to 9 or a functional derivative.
ITRs derived from AAV serotypes AAV1 to AAV12
The first and second ITR sequences are independently derived from an ITR sequence of an AAV serotype selected from AAV1 through AAV12.
Gene expression cassette includes at least one promoter and/or polyadenylation sequence
The gene expression cassette includes at least one promoter and/or a polyadenylation sequence.
Heterologous polynucleotide encodes a therapeutic gene
The heterologous polynucleotide sequence encodes a therapeutic gene.
Across the independent and listed refinements, the claim coverage centers on an asymmetrically modified second ITR with deleted B/B′/C/C′/RBE′ regions and a selected deletion of A or A′, constrained by sequence identity to SEQ ID NOs while maintaining functional trs and RBE activities and requiring an open-end configuration that avoids stem-loop formation.
Stated Advantages
Increases AAV productivity.
Increases transgene expression efficiency.
Reduces genotoxicity by suppressing concatemer formation and reducing genotoxic/integration-associated signals.
Reduces persistence across passages.
Documented Applications
Gene therapy vectors and compositions using the disclosed AAV nucleic acid design.
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