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.
Long-acting cabotegravir (CAB) extends antiretroviral drug (ARV) administration from daily to monthly. However, dosing volumes, injection site reactions, and health care oversight are obstacles towards broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18, and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB, and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics (PK), and biodistribution. PK studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. The NM2CAB, compared to NMCAB and NM3CAB, demonstrated prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg/kg intramuscular injection. These prodrug modifications could significantly improve CAB’s effectiveness.
MABC synthesis and encasement in polymeric nanoformulations improved intracellular drug accumulation and demonstrate translational potential as part of a long-acting antiretroviral regimen.
Long-acting nanoformulated antiretroviral therapy (nanoART) that target monocyte-macrophage could improve the drug’s half-life and protein binding capacities while facilitating cell and tissue depots. To this end, ART nanoparticles that target the folic acid (FA) receptor and permit cell-based drug depots were examined using pharmacokinetic and pharmacodynamic (PD) tests. FA receptor-targeted poloxamer 407 nanocrystals, containing ritonavir-boosted atazanavir (ATV/r), significantly affected several therapeutic factors: drug bioavailability increased as much as 5 times and PD activity improved as much as 100 times. Drug particles administered to human peripheral blood lymphocyte reconstituted NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice and infected with HIV-1ADA at a tissue culture infective dose50 of 104 infectious viral particles/ml led to ATV/r drug concentrations that paralleled FA receptor beta staining in both the macrophage-rich parafollicular areas of spleen and lymph nodes. Drug levels were higher in these tissues than what could be achieved by either native drug or untargeted nanoART particles. The data also mirrored potent reductions in viral loads, tissue viral RNA and numbers of HIV-1p24+ cells in infected and treated animals. We conclude that FA-P407 coating of ART nanoparticles readily facilitate drug carriage and facilitate antiretroviral responses.
Antiretroviral therapy (ART) has changed the outcome of human immunodeficiency virus type one (HIV-1) infection from certain death to a life free of disease co-morbidities. However, infected people must remain on life-long daily ART. ART reduces but fails to eliminate the viral reservoir. In order to improve upon current treatment regimens, our laboratory created long acting slow effective release (LASER) ART nanoformulated prodrugs from native medicines. LASER ART enables antiretroviral drugs (ARVs) to better reach target sites of HIV-1 infection while, at the same time, improve ART's half-life and potency. However, novel ARV design has been slowed by prolonged pharmacokinetic testing requirements. To such ends, tri-modal theranostic nanoparticles were created with single-photon emission computed tomography (SPECT/CT), magnetic resonance imaging (MRI) and fluorescence capabilities to predict LASER ART biodistribution. The created theranostic ARV probes were then employed to monitor drug tissue distribution and potency. Intrinsically Indium (In) radiolabeled, europium doped cobalt-ferrite particles and rilpivirine were encased in a polycaprolactone core surrounded by a lipid shell (InEuCF-RPV). Particle cell and tissue distribution, and antiretroviral activities were sustained in macrophage tissue depots. InEuCF-PCL/RPV particles injected into mice demonstrated co-registration of MRI and SPECT/CT tissue signals with RPV and cobalt. Cell and animal particle biodistribution paralleled ARV activities. We posit that particle selection can predict RPV distribution and potency facilitated by multifunctional theranostic nanoparticles.
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