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Assignees
Member
University of Nevada, Las VegasThe University of Nevada, Las Vegas is a public research university offering comprehensive undergraduate, graduate, and professional programs. The institution is recognized for research initiatives, interdisciplinary curriculum, community partnerships, and diverse student support. UNLV provides resources spanning financial assistance, technology, career development, outreach, and research, advancing individual achievement and community engagement in a multicultural environment.
The University of Nevada, Las Vegas is a public research university offering comprehensive undergraduate, graduate, and professional programs. The institution is recognized for research initiatives, interdisciplinary curriculum, community partnerships, and diverse student support. UNLV provides resources spanning financial assistance, technology, career development, outreach, and research, advancing individual achievement and community engagement in a multicultural environment.
Publication Number
US-12385069-B2
Publication Date
2025-08-12
Expiration Date
Abstract
Disclosed are compositions and methods for studying a Tat gene. Specifically, the disclosure provides a vector comprising a double-stranded nucleic acid construct which comprises a Tat gene and a green fluorescent protein (GFP) reporter element, and further wherein the double-stranded nucleic acid construct comprising AAVS1 (adeno-associated virus integration site, a safe harbor locus) arms that flank on both sides of the Tat gene and the reporter element for integration at the human AAVS1 site by homologus recombination. Further provided are methods of using a cell comprising the vector for studying the effects of exogenous conditions on expression of the Tat gene.
Core Innovation
The invention provides a GigaAssay system for studying HIV Tat activity using a vector comprising a double-stranded nucleic acid construct. The construct includes a first strand and a second strand, each carrying AAVS1 locus left and right arm elements and functional sequences positioned to support a safe-harbor integration context. The system is organized so that Tat is associated with a driver element while GFP is associated with a reporter element.
The double-stranded construct includes promoter and coding sequences on opposite strands, including a CMV promoter and a tat cDNA coding sequence on the first strand and a LTR promoter and a GFP coding sequence on the second strand. Each strand further includes a 3′ UTR containing synthetic poly(A) signals and complementary 3′UTR elements that link the strand-specific elements through complementarity. The 3′ UTRs also include barcode sequences that are complementary across strands.
The invention uses complementary barcode duplex organization in the overlapping 3′UTR regions to link driver and reporter transcripts at the single-cell level, enabling assessment of Tat mutant activity. The system conceptually supports creating a heteroallelic AAVS1 cell line, integrating a barcoded cassette library into the AAVS1 safe-harbor locus, applying conditions, and then using reporter-based flow sorting to select cells by GFP while quantifying Tat activity by RNASeq/NGS or qPCR/flow.
Claims Coverage
The provided material contains one independent claim. It recites a specific dual-strand double-stranded nucleic acid vector architecture in which strand-specific AAVS1 locus arms, promoter/coding/UTR elements, and complementary barcode and poly(A) features are arranged to coordinate strand interactions.
Dual-strand AAVS1-arm double-stranded nucleic acid vector with promoter-linked Tat and GFP on opposite strands
A double-stranded nucleic acid construct having a first strand with AAVS1 locus left arm and, on that first strand, a nucleic acid sequence encoding puromycin N-acetyl-transferase, a CMV promoter, and a tat cDNA coding sequence, and a second strand with AAVS1 locus right arm and, on that second strand, a LTR promoter and a GFP coding sequence, with a functional sequence on each strand.
Complementary 3′UTR poly(A)/polySV40(A) features coordinating strand interactions
A first strand 3′UTR comprising a nucleic acid sequence complementary to a synthetic poly(A) signal of the second-strand 3′UTR and a polySV40(A) sequence, and a second strand 3′UTR comprising a nucleic acid sequence complementary to the polySV40(A) sequence of the first-strand 3′UTR and a synthetic poly(A) signal, thereby creating reciprocal 3′UTR complementarity.
Cross-strand complementary barcode sequences in the 3′UTR
A first strand 3′UTR comprising a first barcode sequence complementary to a second barcode sequence of the second strand, and a second strand 3′UTR comprising the second barcode sequence complementary to the first barcode sequence of the first strand, with barcode placement within the 3′UTR region to link strand-specific elements.
Across the independent claim, the inventive coverage centers on a dual-strand, AAVS1-arm vector architecture that places Tat and GFP on opposite strands and uses complementary 3′UTR poly(A)/polySV40(A) and complementary barcode sequences to coordinate strand interactions within the construct.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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