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Targeted cleavage of the HIV-1 genome in human cells using AAV-delivered CRISPR/Cas9

Nickl, Manuela

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Abstract

More than 30 years after the discovery of HIV-1 as the causative agent of AIDS, the disease can still not be cured and is responsible for around 940,000 deaths worldwide in 2017. Antiretroviral therapy, which was started in 1995, rapidly decreased mortality and increased life expectancy. ART is a life-long therapy since treatment interruption leads to rapid rebound of viral loads due to long-lived latently infected cells mainly represented by resting CD4+ T cells. Since ART can have severe side effects and resistant viruses can evolve under suboptimal treatment, there is an urgent need for a cure, which can only be achieved by eradicating the latent reservoir. A promising strategy to do so is to functionally inactivate the latent HIV-1 provirus by mutating different sites of the proviral genome with the help of site-specific designer nucleases. In this work a CRISPR/Cas9 system was established, which is delivered into target cells with AAV vectors and allows simultaneous targeting of three sites of the HIV-1 proviral genome. The gRNAs designed for this purpose were shown to successfully edit the HIV-1 LTRs, pol and gag in a HeLaP4 reporter cell line (HeLaP4-NLtr) with an integrated HIV-1 provirus and in J-Lat T cells, which harbor a latent HIV-1 provirus. Different gRNA combinations were cloned into gRNA multiplexing constructs which allow the expression of up to three gRNAs. These constructs were shown to edit different sites of the provirus simultaneously and facilitate the excision of proviral fragments between different gRNA target sites in HeLaP4-NLtr and J-Lat cells. In J-Lat cells we could additionally show excision of the whole proviral sequence between the LTRs upon treatment with our constructs. Furthermore, the constructs protected HeLaP4 cells against HIV-1 infection and reduced the population of HIV-1 infected cells by up to 80 %. In addition, the amount of released infectious viral particles was reduced by up to 100-fold. To show that our system enables the functional inactivation of latent HIV-1, J-Lat T cells were treated with the gRNA multiplexing constructs and Cas9 followed by transcriptional activation of the provirus. Indeed, viral release was significantly reduced by up to 74 %. To enable the application of our CRISPR system in primary human CD4+ T cells, different AAV serotypes were compared for efficient transduction and AAV6 was shown to enable transduction of up to 60 % of the cells. Nevertheless, pretreatment with our three most effective gRNA multiplexing constructs and Cas9 did not protect the cells against HIV-1 infection, which is possibly caused by low Cas9 expression levels observed in these cells. Collectively, we established a CRISPR/Cas9 system that enables simultaneous editing of up to three sites in the HIV-1 proviral genome, the excision of proviral fragments or of the whole proviral genome, thereby facilitating the protection against HIV-1 infection and functional inactivation of the latent provirus in human cell lines.

Item Type: Dissertation
Supervisor: Kräusslich, Prof. Dr. Hans-Georg
Date of thesis defense: 26 March 2019
Date Deposited: 10 Jul 2019 13:24
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Medizinische Fakultät Heidelberg > Department for Infectiology
Subjects: 570 Life sciences
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