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Efficient Inhibition of Simian Immunodeficiency Virus Replication with Virus Specific Ribonucleic Acid Guided Nuclease Variants.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Efficient Inhibition of Simian Immunodeficiency Virus Replication with Virus Specific Ribonucleic Acid Guided Nuclease Variants./
Author:	Smith, Lisa Marie.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	166 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-03, Section: B.
Contained By:	Dissertations Abstracts International81-03B.
Subject:	Virology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13880039
ISBN:	9781085628730

Efficient Inhibition of Simian Immunodeficiency Virus Replication with Virus Specific Ribonucleic Acid Guided Nuclease Variants.
Smith, Lisa Marie.

Efficient Inhibition of Simian Immunodeficiency Virus Replication with Virus Specific Ribonucleic Acid Guided Nuclease Variants. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 166 p.

Source: Dissertations Abstracts International, Volume: 81-03, Section: B.

Thesis (Ph.D.)--The University of Texas Health Science Center at San Antonio, 2019.

This item is not available from ProQuest Dissertations & Theses.

Acquired immune deficiency syndrome caused by human immunodeficiency virus continues to be a major global health problem. As with human immunodeficiency virus infection in humans, simian immunodeficiency virus infection in rhesus macaques is lifelong due to the establishment of latent reservoir cells that harbor the viral genome integrated into the host genome called a provirus. Clustered, regularly interspaced, short palindromic repeats associated nuclease are ribonucleic acid-guided nucleases that manipulate cellular genomes that have the opportunity of inactivating provirus. Therefore, generating nucleases against simian immunodeficiency provirus with the intent on inhibiting virus production is a highly translatable viral therapeutic for human immunodeficiency virus that can be tested in non-human primate animal models.We developed simian immunodeficiency virus specific ribonucleic acid -guided nucleases and paired nickases targeting seven conserved regions of the provirus. In vitro assays in human embryonic kidney cells co-transfected with plasmid containing provirus and plasmid encoded green fluorescent protein and ribonucleic acid-guided nucleases were used to select the most effective constructs that inhibited virus production. When ribonucleic acid-guided nucleases target the long terminal repeat, the trans-activation response element, or the ribosomal slip site, there is a dramatic inhibition of virus production and induction of mutations at the target sites. Paired nickases targeting these same regions inhibited virus production significantly, but not as robustly as nucleases. We constructed multiplex plasmids that co-express 3 guide ribonucleic acids and nuclease, or 6 guide ribonucleic acids (three pairs) and nickase that target the long terminal repeat, the trans-activation response element, or the ribosomal slip site. The multiplex plasmids suppress virus production even more efficiently than the single or paired nuclease and nickase encoded plasmids.2C11 cells are a clonal isolate of simian immunodeficiency virus infected T lymphoblast cells that produce high levels of virus capsid protein making it a suitable cell line to test the efficiency of our constructs ability to inactivate integrated provirus. 2C11 cells were nucleofected with the ribonucleic acid-guided nuclease and paired nickase multiplexed plasmids, sorted for green fluorescent protein expression, and cultured for 15 days. Inhibition of virus production for the nucleofected ribonucleic acid-guided nuclease or paired nickase multiplexed plasmids in 2C11 cells was 96% and 81%, respectively, compared with non-nucleofected 2C11 cells. Additionally, multiplexed treated cells had observable mutations at target sites. In summary, our multiplex plasmids can inhibit viral production, and this inhibition inversely correlates with the incidence of mutations in the integrated provirus.We utilized non-genome editing inactive ribonucleic acid-guided nucleases to inactivate provirus via transcriptional interference. In vitro assays in human embryonic kidney cells co-transfected with plasmid containing provirus, plasmid encoded green fluorescent protein, and inactive ribonucleic acid-guided nuclease and our trans-activation response element guides exhibited provirus inactivation. Therefore, when targeting the trans-activation response element without generating mutations through deoxyribonucleic acid cleavage, we are able to detect transcriptional interference. Furthermore, we utilized inactive ribonucleic acid-guided nuclease fused to a Kruppel-associated box domain to inhibit proviral transcription by epigenetic modifications in the provirus. Transduced 2C11 cells that express inactive ribonucleic acid-guided nuclease fused to a Kruppel-associated box domain targeting the trans-activation response element have a marked reduction of intracellular capsid protein as assessed by flow cytometry. Additionally, these cells collectively produce fewer viruses, as detected in the supernatant by Luminex assay. Conversely, 2C11 cells transduced with inactive ribonucleic acid-guided nuclease targeting the trans-activation response element do not reduce intracellular capsid protein. Therefore, epigenetic modifications may be more effective in inactivating proviral deoxyribonucleic acid than transcriptional interference when targeting integrated provirus and thus may be a better strategy to inactive provirus in vivo.

ISBN: 9781085628730Subjects--Topical Terms:

642304
Virology.
Subjects--Index Terms:

CRISPR

Efficient Inhibition of Simian Immunodeficiency Virus Replication with Virus Specific Ribonucleic Acid Guided Nuclease Variants.
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245 1 0 $a Efficient Inhibition of Simian Immunodeficiency Virus Replication with Virus Specific Ribonucleic Acid Guided Nuclease Variants.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2019
300 $a 166 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-03, Section: B.
500 $a Advisor: Giavedoni, Luis D.
502 $a Thesis (Ph.D.)--The University of Texas Health Science Center at San Antonio, 2019.
506 $a This item is not available from ProQuest Dissertations & Theses.
506 $a This item must not be sold to any third party vendors.
506 $a This item must not be added to any third party search indexes.
520 $a Acquired immune deficiency syndrome caused by human immunodeficiency virus continues to be a major global health problem. As with human immunodeficiency virus infection in humans, simian immunodeficiency virus infection in rhesus macaques is lifelong due to the establishment of latent reservoir cells that harbor the viral genome integrated into the host genome called a provirus. Clustered, regularly interspaced, short palindromic repeats associated nuclease are ribonucleic acid-guided nucleases that manipulate cellular genomes that have the opportunity of inactivating provirus. Therefore, generating nucleases against simian immunodeficiency provirus with the intent on inhibiting virus production is a highly translatable viral therapeutic for human immunodeficiency virus that can be tested in non-human primate animal models.We developed simian immunodeficiency virus specific ribonucleic acid -guided nucleases and paired nickases targeting seven conserved regions of the provirus. In vitro assays in human embryonic kidney cells co-transfected with plasmid containing provirus and plasmid encoded green fluorescent protein and ribonucleic acid-guided nucleases were used to select the most effective constructs that inhibited virus production. When ribonucleic acid-guided nucleases target the long terminal repeat, the trans-activation response element, or the ribosomal slip site, there is a dramatic inhibition of virus production and induction of mutations at the target sites. Paired nickases targeting these same regions inhibited virus production significantly, but not as robustly as nucleases. We constructed multiplex plasmids that co-express 3 guide ribonucleic acids and nuclease, or 6 guide ribonucleic acids (three pairs) and nickase that target the long terminal repeat, the trans-activation response element, or the ribosomal slip site. The multiplex plasmids suppress virus production even more efficiently than the single or paired nuclease and nickase encoded plasmids.2C11 cells are a clonal isolate of simian immunodeficiency virus infected T lymphoblast cells that produce high levels of virus capsid protein making it a suitable cell line to test the efficiency of our constructs ability to inactivate integrated provirus. 2C11 cells were nucleofected with the ribonucleic acid-guided nuclease and paired nickase multiplexed plasmids, sorted for green fluorescent protein expression, and cultured for 15 days. Inhibition of virus production for the nucleofected ribonucleic acid-guided nuclease or paired nickase multiplexed plasmids in 2C11 cells was 96% and 81%, respectively, compared with non-nucleofected 2C11 cells. Additionally, multiplexed treated cells had observable mutations at target sites. In summary, our multiplex plasmids can inhibit viral production, and this inhibition inversely correlates with the incidence of mutations in the integrated provirus.We utilized non-genome editing inactive ribonucleic acid-guided nucleases to inactivate provirus via transcriptional interference. In vitro assays in human embryonic kidney cells co-transfected with plasmid containing provirus, plasmid encoded green fluorescent protein, and inactive ribonucleic acid-guided nuclease and our trans-activation response element guides exhibited provirus inactivation. Therefore, when targeting the trans-activation response element without generating mutations through deoxyribonucleic acid cleavage, we are able to detect transcriptional interference. Furthermore, we utilized inactive ribonucleic acid-guided nuclease fused to a Kruppel-associated box domain to inhibit proviral transcription by epigenetic modifications in the provirus. Transduced 2C11 cells that express inactive ribonucleic acid-guided nuclease fused to a Kruppel-associated box domain targeting the trans-activation response element have a marked reduction of intracellular capsid protein as assessed by flow cytometry. Additionally, these cells collectively produce fewer viruses, as detected in the supernatant by Luminex assay. Conversely, 2C11 cells transduced with inactive ribonucleic acid-guided nuclease targeting the trans-activation response element do not reduce intracellular capsid protein. Therefore, epigenetic modifications may be more effective in inactivating proviral deoxyribonucleic acid than transcriptional interference when targeting integrated provirus and thus may be a better strategy to inactive provirus in vivo.
590 $a School code: 0853.
650 4 $a Virology. $3 642304
650 4 $a Microbiology. $3 536250
650 4 $a Genetics. $3 530508
653 $a CRISPR
653 $a Genome editing
653 $a HIV
653 $a Nucleases
653 $a SIV
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710 2 $a The University of Texas Health Science Center at San Antonio. $b Microbiology & Immunology. $3 3549161
773 0 $t Dissertations Abstracts International $g 81-03B.
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791 $a Ph.D.
792 $a 2019
793 $a English
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