Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice

Abstract

Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.

Reduce and Control: A Combinatorial Strategy for Achieving Sustained HIV Remissions in the Absence of Antiretroviral Therapy

“…Importantly, CRISPR/Cas9 has now been used to successfully edit HIV-1 in in vitro-infected primary cells and in PBMCs from ART-suppressed individuals [225]. Multiple studies have also shown the successful application of CRISPR-Cas9 in HIV-1 infected humanized mice [226][227][228][229]. These developments culminated in an interesting humanized mouse study showing that the combination CRISPR/Cas9 and a long-acting slow-effective release ART (LASER ART) achieved apparently complete viral clearance in the absence of any detectable Cas9-mediated off-target effects [229].…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…A number of combinations have been investigated, such as targeted integration of an antiviral inhibitor-encoding gene into CCR5 locus [252], simultaneous targeting the entry receptor and viral essential genes [253], and combining CRISPR/Cas9-mediated excision of provirus with ART [254]. This, in some way, resembles the principle of ART that uses a cocktail of medications blocking different steps of HIV-1 replication.…”

Meeting report: 32nd International Conference on Antiviral Research

“…Howard Gendelman focused on the potential use of long-acting antiretrovirals as a step towards improving treatment outcomes, highlighting some recent advances (Dash et al, 2019).…”

“…Howard Gendelman introduced the concept of long-acting slow effective release antiretroviral therapy (LASER-ART) (Dash et al, 2019), that is, hydrophobic lipophilic nanocrystals that serve as agents capable of slowly releasing antiretrovirals prodrugs. The antiretrovirals are modified to improve drug potency, enhance cell membrane permeability, and facilitate encapsulation into nanocrystals that may be rapidly taken up by cells and distributed into the target tissues (Gendelman et al, 2019).…”

“…Howard Gendelman introduced the concept of long-acting slow effective release antiretroviral therapy (LASER-ART) (Dash et al, 2019), that is, hydrophobic lipophilic nanocrystals that serve as agents capable of slowly releasing antiretrovirals prodrugs. The antiretrovirals are modified to improve drug potency, enhance cell membrane permeability, and facilitate encapsulation into nanocrystals that may be rapidly taken up by cells and distributed into the target tissues (Gendelman et al, 2019). One of the examples presented was the nanoformulation of cabotegravir, a HIV integrase inhibitor, that has been packaged into stable particles with high drug loading capacity and capable of targeting the monocyte-macrophages (Zhou et al, 2018) susceptible to HIV infection.…”