Plasmids
Mammalian expression vector pcDNA3.1+ was used to express proteins ZFP362 ZFP362-DNMT3A or ZFP362-DNMT3A derived fusion constructs. For creating pcDNA-ZFP362-DNMT3A, the truncated and fusion constructs were ordered as gBlocks® (Integrated DNA Technology) and cloned into a pcDNA3.1(+) mammalian expression vector, digested with AfeI and Acc65I, using the NEBuilder® HiFi DNA Assembly Master Mix according to the manufacturer’s instructions (New England Biolabs). EXOtic device plasmids pDB60 (booster), pDB68 (Connexin43 S368A), pSA465 (CD63-L7Ae) and pSA462 (nanoluc-C/Dbox) was a kind gift from Dr Martin Fussenegger.21. The C/Dbox was introduced in the 3’UTR of ZFP362 and ZFP362-PAMt digested with Acc65I and NotI through standard oligo cloning methods. The following reagent was obtained through the NIH HIV-1 Reagent Program, Division of AIDS, NIAID, NIH: Human Immunodeficiency Virus 1 (HIV-1-1) NL4-3 BaL Infectious Molecular Clone (p81A-4), ARP-11440, contributed by Dr. Bruce Chesebro55. The sequences of all gBlocks, oligos, and vectors are provided in Supplementary Data 1.
Cell culture and transfection
The HEK293T and immortalized Mesenchymal stem cells (MSC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (GeminiBio) and incubated at 37 °C and 5% CO2. Immortalized MSCs expressing human Telomerase Reverse Transcriptase with basal levels of GFP and Firefly luciferase were a kind gift from Dr. Glackin31. Jurkat cells were infected with 0.01 MOI of pNL4-3-derived HIV-1 to create chronically infected Jurkat cells57. The chronically HIV-1-infected Jurkat (CHI-Ju) were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Thermo Fisher Scientific) with 10% FBS. CHI-Ju were electroporated using the 10 µL Neon™ transfection system (Thermo Fisher Scientific) as per manufacture’s instruction under the following conditions: 1,325 V, 10 ms, 3 pulse. Peripheral Blood Monocytic Cells (PBMCs) were isolated using Histopaque®-1077 (Millipore Sigma) from donated blood and cultured in RPMI in the presence of IL-2 (100 units/ml) (Millipore Sigma catalogue number 11147528001) and 10% FBS. Microglial cells HC69.5 were a kind gift from Dr. David Alvarez-Carbonell, maintained in BrainPhys™ media (Stemcell technology catalogue number #05791)58 and were electroporated using the 10 µL Neon™ transfection system (Thermo Fisher Scientific) as per manufacture’s instruction under the following conditions: 1100 V, 50 ms, 1 pulse.
Collection, purification and cellular exposure of exosomes
In a 10 cm dish, 4 × 106 HEK293T cells were seeded and 24 h later transfected with 24.5 µg total of EXOtic device plasmids (14 µg of nLuc-C/Dbox, or ZFP-C/Dbox, or ZPAMt-C/Dbox, and 3.5 µg each of the pDB60, pDB68 and pSA465 vectors). After 6 h of transfection, the cell culture medium was replaced with exosome-depleted FBS (Thermo Fisher Scientific). After 72 h of transfection, the media was collected and subjected to sequential spins: a low-level spin at 800 × g for 5 mins to remove cells, followed by 2000 × g for 20 mins to remove of cell debris and then subjected to ultracentrifugation performed at 100,000 × g for 2 h. The final precipitant was resuspended in sterile PBS and passed through a 0.22 micron Ultrafree® Centrifugal Filter Units (Millipore Sigma). The size distributions and an absolute number of exosomes were determined using the NanoSight NS300 (Malvern). For all in vitro experiments, an exosome to cell ratio of 104 exosomes per cell was maintained.
RT-qPCR from cells and exosomes
RNA was isolated from CHI-Ju cells (5 × 105) or exosomes (1 × 1010) using the Maxwell® automated simplyRNA isolation kit according to manufacturer instructions (Promega). Equal amounts of RNA were used for Luna® Universal One-Step RT-qPCR Kit (NEB) according to the manufacturer’s instruction. Gene-specific primers sequences are listed in Supplementary Data 1. Roche® LightCycler 96 was used to perform the RT-qPCR and results were analyzed using the LightCycler 96 software (Roche). The PCR conditions were as follows: reverse transcription at 55 °C for 10 min; initial denaturation, 95 °C for 3 min; denaturation, 95 °C for 30 sec; annealing, 60 °C for 20 s for 35 cycles. For calculation of viral copy number/cell as done by Shan et al.22, the equal number of cells were seeded and 100 ml of cell culture supernatant was harvested to isolate viral RNA using Maxwell® RSC Viral Total Nucleic Acid Purification Kit (Promega). These RNA samples along with RNA standard (AcroMetrix™ HIV-1 Panel) were subjected to RT-qPCR for the Gag region, the standard curve was plotted and viral RNA copy/ml was deduced.
Negative staining transmission electron microscopy
Exosomes were absorbed onto glow discharged carbon coated 200 mesh EM grids followed by conventional negative staining with 1% (w/v) uranyl acetate. Images were collected using an FEI Tecnai 12 transmission electron microscope (Thermo Fisher Scientific) equipped with a LaB6 filament and operated at an acceleration voltage of 120 kV. Images were recorded with a Gatan 2 k × 2 k CCD camera (Gatan, Inc.) using Gatan Microscopy Suite software GMS3 at a magnification of 30,000 × and a defocus value of ~1.5 μm59.
Western blotting
Lysate from cells was prepared by resuspending the cells in Mammalian Protein Extraction Reagent (M-PER) (Thermo Fisher Scientific) buffer and protease inhibitor cocktail (Thermo Fisher Scientific) and subjecting them to sonication for 5 cycles of 30 s on and 30 s off. The same protocol was used for making lysates from exosomes suspended in 1% SDS containing RIPA buffer (50 mM Tris HCl, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) Sodium Deoxycholate, 1.0 mM EDTA, 1% (w/v) SDS and 0.01% (w/v) sodium azide at a pH of 7.4). Protein samples were resolved on 10% SDS–PAGE gels (Bio-rad) and transferred onto PVDF membranes using Trans-Blot Turbo Transfer System (Bio-rad). The membranes were blocked with 5% non-fat milk (Millipore Sigma) in Tris-buffered saline (TBS) solution and incubated with primary antibodies in TBS with 0.05% Tween 20 (Millipore Sigma) (TBS-T) overnight at 4 °C. After washing, the blots were reacted with secondary antibodies for 45 min and developed using the enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific). Anti-Myc antibody (Cell Signalling. catalogue no. 9B11), anti-Alix mouse monoclonal antibody (Cell Signalling, catalogue no. 3A9), anti-CD63 mouse monoclonal antibody (Santacruz Biotechnology, catalogue no. SC5275), anti-GAPDH mouse monoclonal antibody (Santacruz Biotechnology, catalogue no. SC47724), anti-Tsg101 mouse monoclonal antibody (Santacruz Biotechnology, catalogue no. SC7964), anti-tubulin rabbit monoclonal antibody (catalogue no. abcam, ab6046). All primary antibodies were used at a dilution of 1:1000. Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Biorad catalogue no. #1706516) or Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (Biorad catalogue no. #1706515) was used at 1:4000 dilution in 1% non-fat milk (Millipore Sigma) in TBS-T.
Chromatin Immunoprecipitation (ChIP) and Methyl DNA precipitation assay (meDIP)
For ChIP or meDIP assay, after four days of transfection with empty vector, ZFP, or ZFP fusion constructs in CHI-Ju cells, 2.5 × 106 cells were harvested for each sample and subjected to the protocol as described previously60. Briefly, cells were cross-linked, quenched, and lysed. The cell lysate was sonicated with a Bioruptor (Diagenode) using pulse interval 30 s ON and 30 s OFF for a total of 30 cycles. For the ChIP assay, 5μl Pierce™ Anti-c-Myc Magnetic Beads (Thermo Fisher Scientific) per 2.5 × 106 cells, were added to the mixture and after incubation at 4 °C for 2 h, beads were washed with a series of buffers with increasing salt concentrations. Finally, DNA was eluted and reverse cross-linked and purified with QIAquick PCR Purification Kit (Qiagen) and the relative enrichment of Myc tagged ZFP or ZFP fusion constructs was determined at the HIV-1 LTR using qPCR. Primer sequences are listed in Supplementary Data 1.
For the MeDIP assay, CHI-Ju cells were harvested 10 days after transfection and subjected to the same treatment as above until after the sonication step. An antibody against 5-methylcytosine (5-mC) (Abcam catalogue no. ab10805) or non-specific IgG was added to the cross-linked and sonicated lysates (1 mg antibody per 107 cells). After antibody incubation overnight, ChIP-grade Protein A/G magnetic beads (Thermo Fisher Scientific) were added for 2 h. Beads were washed in low and high salt buffers60 and proceeded as per ChIP protocol.
After ChIP or meDIP, DNA was subjected to qPCR assay to calculate fold enrichment using Luna® Universal qPCR Master Mix (NEB). Ct values for input samples were adjusted for dilution and fold enrichment was calculated as 100*2^(Adjusted input – Ct (IP)).
Lentiviral plasmid construction and lentivirus production
The exosome producer third-generation Lentivirus plasmid was custom built by VectorBuilder. Inc., which contained a hPGK1 promoter driving expression of CD63-L7Ae and Connexin43 S368A and EF1α promoter driving ZFP362-PAM- C/Dbox expression. The inclusion of protease cleavage site P2A between CD63-L7Ae and Connexin43 S368A ensured protein separation. Mutations in the bovine growth hormone polyadenylation (bgh-PolyA) transcription termination signal after CD63-L7Ae and Connexin43 S368A enable read-through and expression of complete lentiviral genomic RNA expressed from the RSV promoter. The faulty poly (A) signal allowed for CD63-L7Ae and Connexin43 S368A expression. To obtain pLV-EXOtic-ZFP, DNA fragment between Afe1 and Acc65I i.e., PAMt was removed, overhangs filled in using Klenow Fragment (NEB) and re-ligated. To build the pLV-EXOtic-nLuc vector, the pLV-EXOtic-ZPAMt vector was digested with NsiI and BstBI (NEB) to remove the ZPAMt and replaced with the nLuc gene using NEBuilder HiFi DNA Assembly Master Mix (NEB) according to the manufacturer’s instructions. The calcium phosphate method (Takara) was used to transfected HEK 293 T cells with a mixture of genome transfer plasmid and packaging plasmid: pRRE, pCMV.Rev, pMD2.G61, at a ratio of 4:2:1:162. The lentivirus vector was used at an MOI of 5 to transduce Mesenchymal Stem cells via spinoculation.
Mice
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (JAX stock #005557, Jackson Laboratory) were maintained in accordance with the Guide for the Care and Use of Laboratory Animals and were housed in Specific Pathogen Free conditions. Animal housing follows a 12 h light/12 h dark cycle, wherein the temperature is maintained between 68–79 ˚F and humidity was maintained between 30–70 percent. All experiments were performed according to the guidelines of the Institutional Animal Committee of the Beckman Research Institute of the City of Hope.
Exosome biodistribution
Nanoluc exosomes were administered into NSG mice in a total of 100 µl volume via RO injection. Four hours after exosome injection, mice were euthanized using an overdose of isoflurane anesthesia. Brain, bone marrow, liver, and spleen were harvested in PBS with a 1X protease inhibitor cocktail (Thermo Fisher Scientific). Organs were homogenized and rinsed with ACK lysis buffer (Thermo Fisher Scientific) before resuspending them in 1 ml of PBS. Nano-Glo® Luciferase assay system (Promega) was used to determine the luciferase activity in the organ lysates as per the manufacturer’s instructions. One hundred microliters of lysate were added to 100 µl of Nano-Glo® luciferase assay buffer and mixed well before adding 1:25 prediluted Nano-Glo® luciferase substrate and luciferase activity detected on the Promega GloMax Discover Microplate Reader Detection System with GM3000 Software (Promega).
Transplantation of HIV-1 infected PBMC in NOD/SCID/IL2rnull mice (hu-NSG)
Development of the hu-PBMC NSG mouse model was described in63. PBMC were cultured in RPMI containing 10% heat-inactivated FBS and IL-2 (100 U/ml) and activated overnight using 25 µl/ml CD3CD28 activator (Stemcell ImmunoCult #10990). Activated PBMCs were infected with 200 ng p24 equivalent of pNL4-3 BAL derived HIV-1 in the presence of 2 µg/ml polybrene. After two days of infection, 1 × 106 activated, infected PBMCs were mixed with 9 × 106 uninfected PBMCs. A total of 1 × 107 PBMCs resuspended in 200 µl of sterile PBS were injected IP into 6 to 8-week old NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wj/SzJ (NOD/SCID/ IL2rnull [NSG]) obtained from Jackson Laboratories. Mice were bled by retro-orbital bleeding and plasma viral loads were analyzed periodically RT-qPCR as described later. Procedures involving HIV-1 or HIV-1-infected mice were performed under general anesthesia.
Establishment of hu-CD34+NSG Model
Engraftment of purified human CD45 + HSC in NSG pups and their HIV-1 infection was done as described elsewhere35. For preparation and engraftment of hu-CD34 cells, human CD34 + HSCs were isolated from fetal liver and thymus tissue obtained from Advance Bioscience Resources following regulatory guidelines. The fetal liver tissue (16–24 weeks of gestation) was treated with collagenase digestion, followed by filtration through a sterile 70 μm nylon mesh filter. Immunomagnetic enrichment for CD34 + cells were performed using EasySep™ Human Cord Blood CD34 Positive Selection Kit (Catalog #18096) (Stem Cell Technologies, Vancouver, BC), per the manufacturer’s instructions. This kit gave us >90% pure CD34 + population, as measured by flow cytometry analysis using an anti-CD34 antibody. For engraftment, a modified intrahepatic injection technique was used for the engraftment of neonatal pups within 72 h of birth. A custom-made Hamilton 80508 syringe/needle was used for the injections (30-gauge, 15 mm long needle with a bevelled edge attached to a 50 μL glass syringe). The maximum volume used for injection with this needle/syringe was 30 μL per pup. Animals were pre-irradiated with 100 cGy 137Cs then allowed to recover in the home cage with heat support for 4–6 h before transplantation. Pups were anesthetized by hypothermia anesthesia, then transplanted with 1.0 × 106 CD34 + cells each by intrahepatic injection. 10–12 weeks after transplantation, blood was collected, and the engraftment was verified using multi-parameter flow cytometry analysis.
Antiretroviral therapy and exosome dose administration
Infected mice with detectable viremia were treated orally for two weeks with cART composed of drugs that block new infections. The cART regimen consisting of Truvada® [tenofovir disoproxil fumarate (TDF; 300 mg/tablet), emtricitabine (FTC; 200 mg/tablet) (Gilead Sciences)] and Isentress® [raltegravir (RAL; 400 mg/tablet) (Merck)], scaled down to the equivalent mouse dosage using the appropriate conversion factor, was administered in a drinking water formulation (sweetened water gel, Medidrop® Sucralose, ClearH20). For 200 ml Medidrop®, ½ Truvada tablet, and ½ Isentress tablet were crushed to powder, mixed by vigorous shaking, and changed weekly as per doses calculated previously64. Mice were bled by retro-orbital bleeding, and peripheral blood cell populations and plasma viral loads were analyzed periodically using RT-qPCR. The oral cART regimen was withdrawn after two weeks and exosome treatment was initiated. Exosomes derived from HEK293T cells packed with nLuc, ZFP, or ZPAMt were retro-orbitally injected at a concentration of 100 × 109 in 100 ul sterile PBS once a week for 6 weeks.
Viral load calculation using RT-qPCR
Serum obtained from retro-orbitally collected blood was subjected to automated RNA isolation using Maxwell® RSC Viral Total Nucleic Acid Purification Kit according to the manufacturer’s instructions (Promega). Viremia was assayed using TaqMan® Fast Virus one-step reverse transcriptase real-time PCR (Thermo Fisher Scientific) with an automated CFX96 Touch Real-Time PCR detection system (Bio-Rad). Copies/ml of HIV-1 RNA was calculated using AcroMetrix™ HIV-1 Panel copies/mL (Thermo Fisher Scientific #950470). The LODs of the mice plasma were found to be around 200 RNA copies/ml under these experimental conditions. The mice were bled and assayed for the rebound of plasma viremia and peripheral cell composition periodically until euthanized. Before euthanizing, under anaesthesia, mice were bled by cardiac puncture to collect 700 μl of blood. After euthanizing, spleen and bone marrow (BM) were harvested from all the mice. Lysates from the spleen and BM were treated with ACK lysis buffer (Thermo Fisher Scientific) and washed with PBS before adding homogenization buffer from Maxwell® RSC simply RNA Kits (Promega) to 1 × 106 cells. RNA was isolated and HIV-1 Gag RNA expression was measured from equal amounts of RNA using Luna® Universal One-Step RT-qPCR Kit (NEB) using HIV-1 Gag region and β-Actin specific primers. From the formalin-fixed paraffin-embedded brain tissues of hu-CD34 + NSG mice, RNA was extracted using Maxwell® RSC RNA FFPE Kit (Promega) as per the manufacturer’s protocol. Taqman probes and primers used for Gag region and bActin PCR are listed in Supplementary Data 1.
MeDIP-qPCR from mice samples
MeDIP from bone marrow samples from all the HIV-1-infected huNSG-PBMC mice was carried out using the EpiQuik MeDIP Kit (Epigentek) as per the manufacturer’s instructions. This kit included a ChIP-grade anti-5-methylCytosine (5mC) antibody and normal mouse IgG for negative control. Briefly, DNA from bone marrow was extracted and sonicated using Bioruptor (Diagenode) for 5 cycles using pulse interval 30 s ON and 30 s OFF, shearing into 200–1000 bp fragments. An aliquot of each sample was set aside as input control, while the remaining portion was subjected to immuno-precipitate with an anti-5mC antibody or normal mouse IgG (1 µg per 2.5 × 105 cells) provided in the kit. DNA was released from the antibody-DNA complex by proteinase K and purified through the specifically designed Fast-Spin Column provided in the kit. Eluted DNA was amplified with primers specific for HIV-1 LTR encompassing ZFP binding site and H2B1A (Supplementary Data 1) with PCR conditions as above.
Toxicity assays
After 10 weeks of infection and therapy regimen, mice were euthanized, and total blood was harvested. Blood was spun at 10 min at 1000–2000 × g using a refrigerated centrifuge to collect plasma and 20 µl of plasma from each mouse in duplicate was used to determine ALT and AST activity using the Alanine Transaminase Colorimetric Activity Assay Kit or Aspartate Aminotransferase Colorimetric Activity Assay Kit according to the manufacturer’s instructions (Cayman Chemical).
Flow cytometry
Samples of peripheral blood were collected by retro-orbital bleeding under general anesthesia and stained for 30 min with 2 µl BUV395-conjugated anti-human CD45 antibody (BD Biosciences, BDB563792), 2 µl BV711-conjugated anti-human CD3 antibody (BD Biosciences, BDB563725), and 5 µl APC-conjugated anti-human CD4 antibody (BD Biosciences, BDB555349). (Stained peripheral blood samples were then lysed with red blood cell lysis buffer and absolute cell counts calculated using BD Liquid Counting Beads (BD Biosciences 335925,). Flow cytometry was performed using a BD Fortessa II instrument (BD Biosciences) and analyzed with the FlowJo software (BD Biosciences).
Immunohistochemistry (IHC)
Mouse brains were fixed in 10% formalin post-necropsy. Fixed brain samples were processed by the Veterinary Pathology Core (City of Hope) into three coronal brain regions, namely levels I (Bregma 1.7-0.14), II (Bregma −2.18 to −2.8), and III (Bregma −5.27 to −7.08). Ten million chronically infected Jurkat cells were washed 2X with PBS, before fixing in 10% formalin for 10 mins. Cells were processed into a paraffin-embedded cell block. Processed and fixed samples were subsequently used for immunohistochemistry (IHC). IHC was performed on Ventana Discovery Ultra IHC automated Stainer (Ventana Medical Systems, Roche Diagnostics). Briefly, formalin-fixed paraffin-embedded tissue blocks were sectioned at a thickness of 5 μm and put on positively charged glass slides. The slides were loaded onto the machine; deparaffinized, rehydrated, endogenous peroxidase activity inhibited, and subjected to antigen retrieval. The slides were then incubated with a primary antibody followed by DISCOVERY anti-Rabbit HQ and then subjected to a DISCOVERY anti-HQ HRP detection system. The stains were visualized by a DISCOVERY ChromoMap DAB Kit, and counterstained with hematoxylin (Ventana), and covered with a coverslip. The primary antibody used was an HIV-1 p24 (NBP2-89962; Bio-Techne, clone 002) diluted 1:2000 for IHC. Slides were scanned by the Light Microscopy Core (City of Hope) on a Hamamatsu Slide Scanner at a 20 X magnification. To analyze the IHC slides, we used QuPath version 0.2.24 (The University of Edinburgh, UK).
Ethics statement
All animal care and procedures have been performed according to protocols reviewed and approved by the City of Hope Institutional Animal Care and Use Committee (IACUC) held by the principal investigator for this application (John Burnett and Kevin Morris, IACUC 16095). The research involves blood specimens from anonymous human subjects with no Health Insurance Portability and Accountability Act of 1996 (HIPAA) identifiers. Discarded peripheral blood from anonymous, healthy adult donors from the City of Hope Blood Donor Center (Duarte, CA) were used for the isolation of PBMCs under a protocol determined by the City of Hope Institutional Review Board (IRB number 19582) to be Not Human Subjects Research under OHRP’s Coded Specimen Guidance. Donors have given their informed consent on the utilization of blood for research purposes on the Blood Donor Center’s Donor Consent Card. Foetal tissue and cord blood units were obtained under a protocol determined by the City of Hope Institutional Review Board (IRB number 17155) to be Not Human Subjects Research under OHRP’s Coded Specimen Guidance and therefore did not require informed consent. These specimens were obtained from commercial specimen vendors with no HIPAA identifiers. Both projects were evaluated by the Institutional Review Board (IRB) of the City of Hope and determined not to involve human subjects research per federal regulation 45 CFR 46.102 (d)(f); IRB#/REF#: 19582/184924 and 17155/141478 and comply with the declaration of Helsinki.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 8.3 software. Data are shown as mean ± SD of triplicates unless otherwise indicated. Statistical analysis was performed using a two-tailed Student’s t-test or one-way ANOVA with post hoc tests, as described. A p value less than 0.05 was designated as statistically significant. No animals were excluded from the analysis.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
