Blog

RF1 Actions 8/21/23

Hello HOPEians,

Here is an update on the RF1 meeting from August 21, 2023. If you have any questions, please reach out to Susana Valente and Doug Nixon.

Actions:

  • Susana Valente – Schedule a meeting with Cedric to go over DUX4 data

  • Roan Lab – Look for DUX4 expression in single-cell RNAseq tonsil datasets (+/- HIV)

  • (In progress) Luisa Mori, Ana Leda, and Susana Valente considering writing a review/summary of all the HIV latency models

Updates:

We heard updates from Quentin Gibaut (Valente Lab) on FUBP3 data,  Qifan Wang (Valente Lab) on  p32 data, and Nick Dopkins (Nixon Lab) on transposable element expression.

Quentin Gibaut (Valente Lab) presented on the host factor FUBP3 promotes HIV transcription. Quentin went into the background of identifying FUBP3 as an HIV transcriptional activator. The chaperone protein p32 was recently discovered to interact with Tat and promote HIV transcription. There are 161 proteins identified through ChAP-MS. ChAP-MS is a new technique to identify proteins involved in the HIV machinery. The goal is to identify the enrichment or loss of proteins in transcriptionally active or silenced HIV promoter loci. The advantages of this are it is selective for HIV and has unbiased detection of multiple proteins associated with the DNA target. FUBP3 was identified by ChAP-MS as an HIV activator.

He shared validation data of FUBP3 as an HIV transcription activator and the mechanistic studies of FUBP3 in the HIV transcription machinery. For future studies, the Valente lab will try to find stimulation pathways and run ChipSeq to share a global overview of the infections. They will try to determine which part of the proteins are required for this, run MD stimulation of FUBP3 to Tat, Chip in JLat -10.6, and the effects of FUBP3 overexpression.

Qifan Wang (Valente Lab) shared the activity of prohibitin-2 in HIV-1 transcriptional regulation and reactivation. He shared data on the prohibitin 2 (PHB2) function. PHB2 expression decreases after HIV infection in primary CD4+ T cells and expression is reduced in the HIV GFP population. PHB2 overexpression inhibits HIV production in 293T cells. PHB2 reduces HIV LTR activity and restricts HIV reactivation in Jurkat 10.6 cells. The preliminary data evidence shows PHB2 plays a negative role in primary CD4+T cell infected with HIV. He shared a paper in Nature, Global landscape of HIV-human protein complexes that shows The PHB2 Co-IPs with Tat.

Qifan summarized that PHB2 expression was decreased during HIV transactivation. CHip-PCR showed PHB2 binding to the LTR and its loss during reactivation. PHB2 OE reduces HIV LTR transactivation driven by Tat and PMA. PHB2 restricts HIV reactivation in Jurkat 10.6 cells. PHB2 binds to tat, likely via the basic and core domains. For future studies, they plan to construct a series of PHB2 deletion variants and deliver them into J-Lat cells with the endogenous PHB2 depleted by shRNAs to define PHB2 domains involved in LTR binding. They will perform RNAseq after PHB2 knockdown in HIV-infected primary CD4+ T cells and study if there is an indirect activity of PHB2 on HIV activity. Lastly, they will perform ChIP studies to confirm the epigenetic outcome of each of the factors recruited by PHB2.

Nick Dopkins from the Nixon Lab shared updates on the transposable element (TEs) expression in the gut microenvironment of people living with HIV (PLWH). The TEs comprise a substantial fraction of the human genome. They looked at the activation of human endogenous retroviruses and its physiological consequences. He shared the paper from Plos Pathogens, Qualitative Differences Between the IFNα subtypes and IFNβ Influence Chronic Mucosal HIV-1 Pathogenesis. The data shows gut biopsies and interferome analysis of gut CD4+ T cells.  TEs are modulated in the gut and influence the HIV status on TE expression in the gut. There is an impact of type 1 interferons on TE expression in gut CD4+ T cells. He shared 6 of his favorite HERV lines that interplay between interferons and HIV status on TE expression in the gut microenvironment.

In summary, they are testing the expression of certain HERV elements, such as LTR19_12p13.31. This is indicative of an overabundant antiviral response observed in the hit microenvironment of PLWH pre-ART. Antigen-presenting mDCs and CD4 populations seem primarily unchanged in the gut. The HERV activity in the gut microenvironment is modulated in PLWH, with TYPE 1 interferons processing shared and differential induction in their expression. The incorporation of HERV activity to interferon-centric studies could explain the heterogeneity of prognostic outcomes in PLWH. In the future, they will work to quantify the expression of differentially expressed TEs in the PBMC data from the same patients and define physiological roles for differentially expressed TEs.

Announcements:

  • The HOPE Collaboratory-Wide Meeting is on Monday, Sept 25th at 9am PT/ 12pm ET

  • The next RF1 standing meeting will be Oct 16th, 2023

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF3 Actions 8/7/23

Hello HOPEians,

Please find below the action items from the RF3 meeting on August 7, 2023. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar.

New Actions

  • Yaping – send instructions to Fran for transferring plasmid

  • Ulrike-  send Martin’s GFP work to Yaping

  • Ulrike – meet with Ott lab on problems with the constructs (this week) and send Jurkat line (being selected for expression)


Actions:

  • Fran and Ulrike meet to discuss JLats updates (in progress)

  • Niren is cutting Rubens AAV and mRNA and will do one round of cells experiments (in progress)

  • Niren – Combine and put a PEG coding on the PLGA, use conjugation coding, and do this at the beginning. Try more extensive pegylation

  • Kumar Lab – Send the Ott lab the mutated sequences (Sequencing clones in Jurkats, VLPs to express base editors that can mutate and fight the response) Ursula Schulze-Gahmen can test for TAR structures

  • Kumar lab – Test if you can use dead Cas9 for base editing

  • Kumar lab- Connect with Ursula for biochemistry for host gene targeting

  • Kumar lab to test LNPs from Niren – Transfection of primary human t cells with aptamer target LNPs with heart tissues, testing in vitro. Testing combination first. (in progress)

  • Ott and Kumar lab – Send Ulrike the constructs to test. Schedule a call to test this

  • Michael – Share mice tissue flash freezing protocols with Priti

    • Priti can test materials sent

  • Lish – work on MTA for this (Brec, latency models)

  • Bring Esper onto RF3 to address their global solutions concerns


Updates:

Niren shared updates on his most recent LNP data. They were able to transfect hematopoietic stem cells in vitro and in vivo.  The macrophage and hepatocyte populations were readily transfected; however, more optimization needs to be done in primary T cells (both in vitro and in vivo). The Murthy Lab gave LNPs with one injection in the humanized mouse model but didn’t see human cells transfected, only murine macrophages and other mouse cells. Melanie has been communicating with Gladstone about collaboration with the Kumar Lab to use non-HIV preliminary work (Rahul will work on this). We will hear updates from Niren on his AAV and mRNA cell experiments.

Fran gave updates on delivery and BrecOFF data. The Ott Lab is trying to optimize the delivery of BrecOFF to mice by molecularly changing the gene to see if they can detect it in another way. He shared a poster from Yaping to demonstrate the “T-cells nucleofection optimization” using Buffers SE, SF, and SG. Of the three buffers and programs, the Buffer SG program is the best but still induced apoptosis in cells.  The GFP control works the best but the cell death is still high. Yaping has been using a new protocol to transfer plasmid and will send instructions to Fran. They will check the clones that were created with similar expressions of BrecOFF and BrecOFF variants. Fran shared a graph of the BrecOFF variants repressing HIV LTR transcription.

Yaping has been working on TAR editing in the ACH2 cell line There is a G:A mutation and it mutated 100% in the two places as confirmed by sequencing. The Kumar Lab wants to see if they can prevent a replication after using the PMA and Ionomycin. Ulrike will send Yaping, Martin’s GFP work. AAV9 is also in the CRISPR-Cas9 recent paper. They modified the AAV to carry the CD4 nanobody to get better expression in lymphocytes.

Ulrike generated some data on CD7 on AAVs. Michael Corely has the results of the CPG methylation on JLat models. Ulrike will share activity on different DNA methylation levels and meet with Melanie about sending a Jurkat line. She can package the AAVs and do a similar co-transfection with a reporter that expresses luciferase.


Announcements:

  • Check out the Community Jam Board to help address some of our community’s concerns

  • The next HOPE Collaboratory-wide meeting is Sept 25, 9am PT/12pm ET.

  • The next RF3 meeting is October 2nd, 2023 and we will hear updates from Niren on his AAV and mRNA cells experiments.

  • Upcoming Speakers:

    • August 21st – Dr. Yasuhiro Arimura

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF2 Actions 8/2/23

Hello HOPEians,

Please find below the action items from the RF2 meeting on August 2nd, 2023. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu. 

New Actions:

  • Roan Lab – Send Valenate Lab tonsil samples (after they determine the best concentration of Tat degraders to use in our system)

  • Ndhlovu Lab – Send material to Roan/Valente lab from the first set of in vivo SP Africa cohort (in progress). Scott and Lish will send more details on the control.

  • Michael Corely – shared paper with RF2 (Ashley’s talk on CD4+ T Cells upregulated expression of Viral sensors post-infection)

Old Actions:

  • Valente Lab – Send XPB protein or plasmid to Paulo Augusto to use as a control

  • Michael Corley- Send Valente lab samples to test the Western blot. Send lysis buffer recipe to test so that DNA, RNA, and protein can be extracted from the same sample)

  • Andrew McAuley – Work with the Brazil team to optimize the XPB Western blot

  • Ndhlovu Lab – create an inventory of received patient samples and create a list of how many cells will go into each test for a systematic analysis 

  • Matthew Parsons – Send nonhuman primate studies/ PK experiment plan to the group soon

  • Valente Lab – Give DNA to Lish to look at the methylation state. 

Updates:

We heard roundtable updates from the Valente, Roan, and Ott labs. 

Nadia and Susana shared brief updates on testing Tat degraders. 

They have been testing at 10 micromolar, however, this is lower than what Valente’s lab would use. The Roan lab has been using a range of 10-100 and looking at the PBMC stimulation model. They will send tonsil samples to the Valente lab once they find an ideal condition. 

Rubens Almeida-Tavora shared data on drugs in combination and adding inhibitors. 

There are ongoing efforts to “block and lock” the HIV promoter. They are looking at the SP+dCA combination studies. The hypothesis is that the initiation and elongation of transcriptional inhibitors together may promote a different epigenetic signature at the HIV locus. He shared a graph of qPCR analysis of Jlat-10.6 cells that are fully suppressed after ~100 days of SP+dCA drug treatment. Spironolactone is typical with the flickering in viral levels, but the dCA is not the best in this case. Due to the difficulty in synthesizing new batches of dCA, they have been using old aliquots of dCA with decreased efficiency. Generally, they split the cells in three days and add fresh drugs each time. Once the drug is removed, the virus rebounds upon dCA and SP interruption but not dCA+SP interruption. The viral setpoint after treatment interruption is still being established. His graph shows drug interruption on day 327. Rubens shows the western blot analysis where SP reemerges as soon as three days after treatment interruption. The Valente Lab is analyzing the western blot 15 days after treatment interruption, unstimulated vs stimulated with TNFa. In addition, they are interested in exploring the combination of the Tat degraders TTX-881 and SP in reducing HIV transcription in j-lat 10.6 (also looking at TTX-856, etc.). They are limited by half-life of the drug in humanized mice, so eventually testing with nonhuman primates can help to extend this.
Lish will send material to Roan/Valente lab from the first set of in vivo SP Africa cohort. They will look at XPB and RNA and DNA and Scott and Lish will share more details on the controls.

Ashley George went over new tools they learned after the recent IAS conference. 

She shared the viral sensing CyTOF panel. The unique aspect of this panel is that it contains a number of intracellular viral sensors and restriction factors listed. Without infection, they change (including HIV fusion assay before production infection)(active vs latency, intermediate stages unknown for now). Looking at the viral sensing across the HIV replication cycle, they can visualize which vital components in the HIV replication cycle these viral sensors target. The CD4+ T cells upregulated the expression of Viral sensors post-infection (data from in vitro infected model and also data from the Last Gift Cohort). They found that productive infections of CD4+ T cells upregulate the expression of a number of viral sensors, including those we identified previously in the mucosa. The next steps are to treat with PREP and ART and show data over time. Michael will share a paper related to this work. 

Ursula Schulze-Gahmen shared data on screening for inhibitors. 

The in vitro solution FRET assay (HTRF) measures TAR binding to tat-sec. They measure the binding of TAR to the super elongation complex. If TAR binds, they get a FRET signal (also control assay). She shared data on the positive controls and the dose-response for Tat degraders from Valente Lab. The Tat Tyr 26 region involved in TAR binding shows a very affinity (still in the micromolar stage). They will test more when more potent. 

Announcements

  • Check out the Community Jam Board to help address some of our community’s concerns

  • The next HOPE Collaboratory-wide meeting is Sept 25, 9am PT/12pm ET. 

  • The next RF2 meeting is Oct 4, 2023

  • Upcoming Speakers:

    • August 21st – Dr. Yasuhiro Arimura

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include. 

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF1 Actions 6/28/23

Hello HOPEians, 

Here is an update on the RF1 meeting from June 28, 2024.

We heard updates from Ursula Schulze-Gahmen (Ott Lab) on RNA conformational propensities determine cellular activity and Ifeanyi Ezeonwumelu (Roan Lab) on targeting the CD127-expressing tissue reservoir by ADCC in the tonsil model.

Actions:

  • Susana Valente – Schedule a meeting with Cedric to go over DUX4 data

  • Roan Lab – Look for DUX4 expression in single-cell RNAseq tonsil datasets (+/- HIV)

  • (In progress) Luisa Mori, Ana Leda, and Susana Valente considering writing a review/summary of all the HIV latency models

Ursula Schulze-Gahmen 

Ursula presented on “RNA conformational propensities determine cellular activity”. In contrast to previous studies on the role of RNA in cellular processes, this work focuses on a quantitative approach to test if the hierarchical organization holds for the cellular activity of HIV-1 TAR.
In the shared study, they used TAR variants that were designed to probe the effect of conformational energetic penalties on the cellular activity of TAR in HIV transcription.  Conformational penalties include stacking energy to convert the predominantly bent TAR molecules into linear stacked molecules, and the energy to form the triple base between U23 and A27-U23. By modulating the TAR-bulge size and introducing mutations that disrupt the essential base-triple motif, they were able to correlate the resulting energetic penalties with the energetics of transactivation by TAR. The study showed that the relatively rare high-energy conformation of TAR is critical for transactivation and that it is important to go beyond probing the dominant structures in vitro and in vivo to quantitatively measure ensembles that describe the propensities to form a biologically active conformational state.

Ifeanyi Ezeonwumelu

Ifeanyi Ezeonwumelu from the Roan Lab presented on targeting the CD127-expressing tissue reservoir by ADCC in the tonsil model.  Ifeanyi shared the Therapeutic strategy to target the IL-7Rɑ (CD127) by antibody-mediated cell-dependent cytotoxicity.  T cells acute lymphoblastic leukemia (T-ALL) are the most common malignancy in children. He shared finds from the paper “New anti-IL-7Rα monoclonal antibodies show efficacy against T cell acute lymphoblastic leukemia in pre-clinical models”. The antibodies are dependent on cellular cytotoxicity (ADCC) and antibodies bind to antigens on target cells. So can we adapt ADCC to specifically target latent HIV-infected memory cells? Can we target the elimination of cells that are highly susceptible to HIV infection when looking at the gating strategy for characterizing the latently-infected CD127+ cells and target HIV-infected CD127+ cells for AdCC depletion? The anti-hIL-7Rɑ antibody 4A10 facilitated a significant depletion of HIV-1 infected cells ex vivo during 3 independent experiments. The next steps are to include more donors and determine the impact of ADCC depletion on latency reactivation in the HIV-infected HLAC model.

Announcements:

  • Y3 subcontracts are being issued.  All Year 2 invoices were due June 30, 2023

  • The HOPE Collaboratory-Wide Meeting is on Monday, July 10th, 9am PT/ 12pm ET

  • The next RF1 standing meeting will be Aug 21, 2023

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include. 

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF3 Actions 6/5/23

Hello HOPEians,

Please find below the action items from the RF3 meeting on June 5, 2023. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar. 

Actions: (Please let us know if we can cross any of these off the list)

  • Fran and Ulrike meet to discuss JLats updates

  • Kumar Lab – Send the Ott lab the mutated sequences (Sequencing clones in Jurkats, VLPs to express base editors that can mutate and fight the response) Ursula Schulze-Gahmen can test for TAR structures

  • Kumar lab – Test if you can use dead Cas9 for base editing

  • Ott and Kumar lab – Send Ulrike the constructs to test. Schedule a call to test this

  • Niren – Combine and put a PEG coding on the PLGA, use conjugation coding and do this at the beginning. Try more extensive pegylation

  • Kumar lab- Connect with Ursula for biochemistry for host gene targeting

  • Bring Esper onto RF3 to address their global solutions concerns

  • Michael – Share mice tissue flash freezing protocols with Priti

    • Priti can test materials sent

  • Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues

  • Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish

 

Updates:

Rubens Almeida-Tavora: Homing Endonucleases

Rubens shared his work on the use of homing endonucleases to inactive adeno-associated virus transgene expression. They are using the Adeno-associated virus (rAAV) as an in vivo gene therapy platform. rAAV has broad tropism, low immunogenicity, and lack of pathogenesis but can also achieve the long-term expression of transgenes. AAV gene therapy can be used to treat hemophilia A.

Rubens shared a study on the functionally cured “Miami Monkey”.  Rhesus macaques infected with SHIV and AAV delivery of monoclonal antibodies showed little to no SHIV viral load. There are pros and cons to the long-term expression of AAV. It is currently impossible to halt AAV expression in case of adverse events. To inactivate AAV transgene expression, we have turned to Homing Endonucleases.

Homing Endonucleases (HEs) are naturally expressed “selfish” mobile genetic elements found across a wide range of organisms. They promote their own propagation through targeted recombination and have many characteristics that are perfect for our goal. In order to screen naturally occurring HE’s, Rubens and colleagues utilized a luciferase-based platform. They compared the toxicity of their three best HEs and designed inactive endonucleases to serve as negative controls. To help improve their target construct, they will change the location and add more targets.

Rubens shared the paper published in Nature on  “Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes”. The study showed that placing introns within the coding region of a gene can significantly increase transgene expression. Using this technology, Rubens and colleagues were able to make improvements to the target construction by implementing new intron technology for Anil-Y2, Bmol, and Ppol reporters. 

Additionally, they introduced an Anil-Y2 within the OSM signal sequence to further improve the target reporter. This can significantly contribute to transgene inactivation. Rubens explained further the design of mRNA expressing HE, LNP delivered HEs toxicity profile, alternative approaches, and the design of their in vivo experiments.

Other Updates:

We also heard quick updates from Ulrike, Fran, and Yaping. Ulrike shared data from their focus meetings on production for specific AAVs in mice. They are looking for biodistribution and want to boost expression for Brec1. Fran shared the Lenti usable dock system for brec1. The doc-inducible dBrec1 and Brecoff express and bind to HIV LTR. They will introduce Doxo and find out why it is not working in JLats. Yaping is working with primary t-cells and improving efficiency on the CCR5 and TAR base editing. The single base mutation was confirmed from sequencing data and it will be hard to transfect cells CCR14.

In the next RF3 meeting, we will hear more updates from Fran on jLat results and from Yaping on CCR5 and TAR base editing data.

Announcements

  • Check out the Community Jam Board to help address some of our community’s concerns

  • The HOPE Collaboratory-wide meeting is July 24th 9am PT/12pm ET.. 

  • The next RF3 meetings is August 7th

  • Upcoming Speakers:

    • June 15     Dr. Mimi Ghosh

    • June 26     Drs. Steven Deeks & Michael Peluso

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include. 

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF2 Actions 6/7/23

Hello HOPEians,

Please find below the action items from the RF2 meeting on June 7, 2023. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu.

 

Actions:

  • Valente Lab – Send XPB protein or plasmid to Paulo Augusto to use as a control

  • Michael Corley- Send Valente lab samples to test the Western blot. Send lysis buffer recipe to test so that DNA, RNA, and protein can be extracted from the same sample)

  • Andrew McAuley – Work with the Brazil team to optimize the XPB Western blot

  • Ndhlovu Lab – create an inventory of received patient samples and create a list of how many cells will go into each test for a systematic analysis

  • Susana will give a talk about spironolactone to the ACTG

  • Matthew Parsons – Send nonhuman primate studies/ PK experiment plan to the group soon

  • Valente Lab – Give DNA to Lish to look at the methylation state.

 

Updates:

During the RF2 meeting, we went around the table and heard updates from each site. Andrew McAuley shared updates from the last Spironolactone working group. They are working on the XPB western blot and described the best way to preserve the PBMC.

Vivian Avelino-Silva shared some progress on their studies at USP.  They will start collecting samples on Monday. They currently have 40 PLWH that are on spironolactone and will track age, gender, and time with diagnosis. They found 9 patients who currently have detectable viral loads, and 4 patients to start spironolactone so they can get before & after data. Luciferase is incorporated and will be offered as an optional procedure. They started the pilot study with healthy volunteers, measuring XPB to send to the Valente Lab.

Matt Parsons shared updates with MHRP. They are on schedule with their studies, but they are having trouble with sodium chloride solution from the local water in Thailand. They need to optimize before proceeding. They will have preliminary data from their primate study in the Fall.

Betty Mwesigwa with MUWRP in Uganda shared updates on the current political changes regarding Uganda’s new anti-gay legislation with the death penalty in some cases. They will continue their research but are reorganizing their procedures, recruitment, and language their staff use, in order to protect the participants. They plan to hold a meeting with their lawyer to get clear guidelines, as well as a meeting with national stakeholders to discuss the best way to move forward. In the meantime, they hope to enroll two new participants but need more guidance on which samples to prioritize.

Andrew Atkins and Scott Bowler from the Ndhlovu Lab shared data and sample processing flow from the NHS clinical studies, including 63 unique patient PIDs. They need to extract protein from all cells and send them to the Valente lab to test reservoir assay and XPB data while simultaneously extracting DNA and RNA for additional analyses. The PBMC samples have arrived, inventoried, and stored. The other plasma samples arrived and shipment is pending for the remaining.

Sara Gianella Weibel from UCSD shared they have limited amounts of tissue and have initial plans to work with Michael Corley.

Announcements

  • Check out the Community Jam Board to help address some of our community’s concerns

  • The next HOPE Collaboratory-wide meeting is July 24th, 9am PT/12pm ET.

  • The next RF2 meeting is August 2nd

  • Upcoming Speakers:

    • June 15     Dr. Mimi Ghosh

    • June 26     Drs. Steven Deeks & Michael Peluso

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

Collab-Wide Actions 5/22/23

Dear HOPEians,

Thank you for attending the first HOPE Collaboratory-Wide Meeting on May 22, 2023. Below is a summary of the meeting, actions, and announcements.

 

RF2: Daniela Boehm – SMYD5 screen update

Today, Daniela Boehm, from the Ott Lab, shared an update on SMYD5 inhibitor screening. SMYD5 is a lysine methyltransferase and is required for HIV transcription.  SMYD5 methylates histones and Tat in vivo. Activity tests with Tat peptides performed by collaborators at SGC Toronto showed SMYD5 has strong methylation activity at Tat aa38-72. They use promega MTase-Glo ™ Methyltransferase assay to determine the optimal SYMD5 and Tat peptide concentration with titrations. They are screening a library of known MTase inhibitors. Daniela is working on a virtual inhibitor screening with Andrii Kyrylchuk and Brian Shoichet from the UCSF School of Pharmacy. Andrii and Brian compared the SAM binding site in published SMYD family proteins SMYD2 and SMYD3 to the alphafold stucture of SMYD5. Comparing the SAM binding site, the cofactor-binding site showed a well-aligned structure between SMYD5 and other SMYD proteins. They also observed water in the binding site. Andrii and Brian virtually screened ~2 Billion fragments and picked ~200 fragments to screen in the SMYD5 inhibitor screen. Hit fragments will later be chemically modified to develop an inhibitor that is specific for SMYD5.

 

RF1: Qifan Wang- Prohibitin 1 & 2 and HIV transcriptional regulation

Qifan Wang, from the Valente Lab, shared his research on the activity of prohibition-2 in HIV-1 transcriptional regulations and reactivation. There are ongoing efforts to “block and lock” the HIV promoter. They identify novel protein regulators associated with active and latent HIV promoters while utilizing a compound from previous work to make HIV-infected sites latent. Some data showed enrichment from the chromatin affinity purification by CRISPR dCAS9 of transcriptionally active and latent HIV promoters. Quifan showed data from the putative “hit” HIV transcriptional regulators with both enriched active and latent LTR. There is overexpression and PHB2 inhibits HIV production in 293T cells. PHB2 reduces HIV LTR transactivation driven by Tat and PMA. PHB2 seems to bind directly to the HIV promoter and is displaced upon LTR activation with PMA. PHB2 expression is reduced in the HIV GFP+ populations, in HIV+ expressing Jurkat cells, and decreased after HIV infection in primary CD4+T cells. PHB2 restricts HIV reaction in Jurkat 10.6 cells. There is a knockdown of PHB1 also seems to increase HIV reactivation. Quifan shared some insight from the paper in Nature “Global landscape of HIV-human protein Complexes”. PHB2 expresses both in the cytoplasm and nucleus and shows some degree of co-localization. PHB2 binds to tat, likely via the basic and core domains

 

Community: Patricia Defechereux 

Patricia Defechereux shared some community updates, recent Community projects, and positive reactions from the public. Here are some current HOPE community projects to check out:

Tom Villa, one of HOPE’s Ambassadors, has been working with HIV cure clinical trials. In Positively Aware magazine there is a 4-part series featuring first-person accounts from participants in HIV cure clinical trials with Analytical Treatment Interruptions (ATI). Co-present with Karine Dubé at the ACTG Annual Meeting (June 13-16) on work with the ACTG Partner Protection Working Group to mitigate unintended HIV transmission during HIV cure-related clinical trials with ATI. Please check out the attached slides to see a list of upcoming Community events including the C2U Expo in Canada, a participants appreciation event in São Paulo, CAIR FOCUS Groups, etc.

 

RF3: Niren Murthy – LNP Targeting

Niren Murthy at Berkely shared some data from his research on LNP targeting. His primary objective for RF3 is to develop LNP/mRNA complexes that can deliver gene editor mRNA to T cells and HSPCs. Azide acetal is a new linker for generating acid-degradable drug delivery vehicles. LNPs made with acid-degradable lipids should disrupt endosomes efficiently and have lower tissue accumulation. There are too many Peg inhibitors in the function of LNPs. He shared data from LNPs with acid-degradable PEG chains transfect HSPCs in vitro with low toxicity and in vivo with Cre mRNA. For future work, he will examine the effects of GM-CSF and perform bone marrow transplants of transfected HSPCs. Looking at Acid-degradable cationic lipids, they found new lipids can be synthesized rapidly and cheaply via an automated robotic system (ARS). Lipids generated via automated LNP synthesis transfect primary T Cells and peptide lipids transfect the spleen. His next goals are to send LNP/luciferase mRNA to Kumar’s lab for testing in a humanized mouse model; deliver Cas9 mRNA and gRAN for CCR5 in vitro to T cells and HSPCs; and expand the peptide-lipids library.

Announcements

  • Year 3 began May 1st, Please spend down your budget and send invoices

  • Check out the Community Jam Board to help address some of our community’s concerns

  • The next RF meetings: Please come prepared with some updates.

    • RF1   June 28   (Ursula- RNA conformational propensities determine cellular activity)

    • RF2   June 7     (TBD)

    • RF3   June 5     (Rubens- Homing Endonucleases)

  • Upcoming HOPE Guest Speakers:

    • June 26    Steven Deeks, MD & Michael Peluso, MD

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF1 Actions 4/17/23

Hello HOPEians,

Here is an update on the RF1 meeting from April 17th, 2023.

We heard updates from Ana Leda on dCA-resistant viruses, Zichong Li on activating PP2A and inhibiting P-TEFb synergistically block reactivation of latent HIV, and Smitha Srinivasachar Badarinarayan on TRIM22/ZNF579 as silencers of HIV.

Ana Leda- dCA Resistant Viruses

Ana came back to present on her last project in the Valente Lab focusing on the loss of in vivo replication fitness of HIV-1 variants to the tat-inhibitor – dCA. She shared some data from the paper: Resistance to the Tat Inhibitor Didehydro-Cortistatin A Is Mediated by Heightened Basal HIV-1 Transcription. When testing ultra-deep sequences to identify dCA resistance, they found no mutations in Tat or Tar. In collaboration with Dr. Victor Garcia, they used his humanized mouse models to test for in Vivo replication kinetics of dCA-resistance viruses and t-cells evaluated weekly. The wild-type infected animals are more prone to infections. Ana shared data shared on human T-cell levels in peripheral blood and the frequency of activated human T-cells in peripheral blood. dCA-resistant HIV-1 variants established a systemic infection in humanized mice. The hypothesis was dCA-resistant HIV-1 variants do not elicit INF responses. They submitted plasma samples for a Luminex analysis for different levels of cytokines and found no difference between M1 and M2 animals. The ability of dCA-resistant HIV-1 variants to establish infection correlated with reversion to wild-type. In conclusion, they found that in vitro-selected dCA-resistant variants have high replication fitness and elevated cytopathogenicity in vitro they are not fit in vivo. They also found that dCA-resistant viruses identified in vitro suffer a large fitness cost in vivo, with mutations in the LTR and nef regions the most pressure to revert to wild type.

Zichong Li- “Activating PP2A and inhibiting P-TEFb synergistically block reactivation of latent HIV”

Zichong validated using siRNAs the synergies between INTS2, PSMD8, and FTSJ3 identified by CRISPRi screens. The FTSJ3 had proteasomes synergize with an integrator, but not each other. There is a synergistic effect between the two drugs. AZD4573 + SMAP-2 blocks and locks latent HIV in cell lines and a primary cell model. The epigenetics changed by the combination of AZD4573 + SMAP-2 on HIV LTR include increased H3K9me3 and H3K27me3 in the Nuc-1 region. Looking at the cellular toxicity tests for AZD4573 + SMAP-2 he found that one-day treatment is tolerated, but on day two the cells die. In the future Zichong will test new HIV Silencing promoting agents and look for synergy with other SPAs; side effects; and longer locking effects. Zichong found that AZD4573 + SMAP-2 could block HIV reactivation and induce inhibitory epigenetic changes on HIV LTR that can last for at least six days. Among the epigenetic changes are increased levels of H3K27me3, which plays a major role in silencing latent HIV.

Smitha Srinivasachar Badarinarayan -TRIM22/ZNF579 as silencers of HIV

Smitha gave updates and background on TRIM22. TRIM22 is a member of the family of tripartite motif proteins, and interferons induce it, restrict IAV, HCV, HBV, etc. via different mechanisms and inhibit HIV transcription, and promote HIV latency. Feschotte lab observed that TRIM22 binding colocalizes with CTCF genome-wide and a further decrease in CTCF binding in TRIM22-depleted cells. CTCF is a regulator of HIV latency. It is involved in many cellular processes, including transcription regulation, insulator activity, and regulation of chromatin architecture. There was a significant overlap between TRIM22 and CTCF ChIP-seq peak. They found an overlap of ChIP-seq peaks between ZNF579 and TRIM22 genomic bindingFurther, found a predicted binding motif of ZNF579 was within the HIV-1 LTR. They plan to validate TRIM22 findings; co-immunoprecipitation to determine if TRIM22 can bind directly or indirectly to CTCF or other factors; TRIM22 HiC/4C-seq; analyze the effect the ZNF579 and TRIM22 on HIV-1 latency in JLat A2 GFP cell line model; and RNA and protein expression of TRIM22/ZNF579 in HIV-1 infected samples. Preliminary results showed that TRIM22 reduced reactivated HIV-1 in stimulated Jlat GFP cells.

Actions for next meeting:

  • Susana Valente – Schedule a meeting with Cedric to go over DUX4 data
  • Roan Lab – Look for DUX4 expression in single-cell RNAseq tonsil datasets (+/- HIV)
  • (In progress) Luisa Mori, Ana Leda, and Susana Valente considering writing a review/summary of all the HIV latency models

Announcements:

  • Check out the Block, Lock, Excise for HIV Cure animation Here! 
  • The HOPE Collaboratory-Wide Meeting is on Monday, May 22, 9am PT/ 12pm ET
  • The next RF1 standing meeting will be on June 28th
  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF2 Actions 4/5/23

Hello HOPEians,

Please find below the action items from the RF2 meeting on April 5, 2023. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu.

New Actions:

  • Valente Lab – Send XPB protein or plasmid to Paulo Augusto to use as a control
  • Michael Corley- Send Valente lab samples to test the Western blot. Send lysis buffer recipe to test so that DNA, RNA, and protein can be extracted from the same sample)
  • Andrew McAuley – Work with the Brazil team to optimize the XPB Western blot
  • Ndhlovu Lab – create an inventory of received patient samples and create a list of how many cells will go into each test for a systematic analysis

Old Actions:

  • Susana will give a talk about spironolactone to the ACTG
  • Matthew Parsons – Send nonhuman primate studies/ PK experiment plan to the group soon
  • Valente Lab – Give DNA to Lish to look at the methylation state.

Updates:

Susana Valente gave an update on her meeting with Betty Poon and Gerard Lacourciere with the NIH during the Miami meeting to discuss Tat inhibitors. She shared that there are two paths to choose from. One might have more feedback with compounds in collaboration with Southern Research. Alternatively, another company would supply the Valente lab with novel compounds that can be tested to inhibit Tat-mediated transcription. A new chemist at the University of Florida-Scripps will advise when choosing compounds.

Susana also shared that there is a continued problem with dCA synthesis. Their new Chemist is good with the natural synthesis of products and is excited to help improve dCA production. Andrew McAuley from the Valente Lab looked at XPB degradation following spironolactone treatment using various cell types and conditions.  The Valente lab hypothesized that sodium fluoride within the SP-treated samples is creating abnormal results on the western blot (i.e. no control protein -GAPDH/Tubulin bands are showing up) Andrew will continue optimizing to try to overcome this problem and is currently working on a flow-based assay to measure XPB degradation. Ana Carolina and Paulo from the Brazil team showed their XPB western blot results. They are facing other problems with Western blot protocol and working on modifications to get better results. Andrew from the Valente lab will share their optimized protocol with the Brazil group to improve their western blots.

Lish received (estimated 107) samples today from the Natural History Studies cohort.  Their plan is to isolate and analyze the DNA and RNA simultaneously. They can save the protein from those same cells for downstream Westerns or ELISAs. Michael will send Valente lab samples to test by Western blot.

Is there a plan for single-cell RNAseq? Nadia shared that you could test the leftover cells RNA seq on fixed cells using a “fixed scRNAseq kit”. Here is the fixation protocol from 10X. 10X recommends 300K cells before fixation. The fixation kit costs about $25 a sample. You can freeze the cells to test later.

In the next meeting, Scott Bowler will give a complete update on new samples (pre and post-spironolactone tests). We will discuss non-suppressible viremia in ART-treated individuals. We would also like to hear highlights from the Tat degrader projects in the Valente Lab, p32 interaction with Tat, spironolactone updates from the Brazil team, and an update on the Last Gift meetings from Lish and Nadia.

Announcements

  • Check out the Community Jam Board to help address some of our community’s concerns
  • The HOPE Collaboratory-wide meeting is May 22nd 9am PT/12pm ET..
  • The next RF2 meeting is June 7th
  • Upcoming Speakers:
    • April 24     Dr. Jon Karn
    • May 1       Dr. Jeannette Tenthorey
    • May 15     Dr. Steve Yukl
  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

Best regards,


Sydney Norman

HOPE Collaboratory Project Coordinator
Gladstone Institutes | UCSF

RF3 Actions 4/3/23

Hello HOPEians,

Please find below the action items from the RF3 meeting on April 3, 2023. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar.

Updates:

Today we heard updates about Niren’s acid-degradable LNPs data. His project goals are to develop LNP/mRNA complexes that can deliver gene editor mRNA to T cells and HPSCs. LNPs made with acid-degradable lipids should disrupt endosomes efficiently and have lower tissue accumulation. Acid-degradable LNPs transfect a variety of non-liver organs in mice, including the liver, spleen, and heart. LNPs with acid-degradable PEG chains transfect the bone marrow and spleen and HSPCs in vitro with low toxicity. However, myeloid populations appear to be the main transfected population, rather than T cells.  Low doses of LNPs with acid-degradable negative lipids transfect 5% of the CD3+ cells in the spleen. The Murthy Lab confirmed ZsGreen positive cells in BM reproducibly and detected ZsGreen positive cells in peripheral blood cells. They can target ST-HSC in vivo and can detect the ZsGreen signal in almost all hematopoietic lineages.

New lipids can be synthesized rapidly via solid-phase synthesis. Peptide-lipids generated via solid-phase synthesis can transfect primary T-cells. Heavily modified gRNAs increase the gene editing efficiency of LNP-delivered CAS9 mRNA in primary neurons (derived from iPSC). Niren shared the paper: A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

We also heard updates from Martin Hamann on AAV-mediated Brec1 delivery and an overview of their cell models. Brec1 delivered by lentivirus leads to the excision of HIV-BFP reporter. Initially, there is little to no Brec1-mediated recombination seen in any of their reporter lines when delivered with AAV. They tested optimized Brec-1 ORF, exchanged promoters, and utilized multiple AAV serotypes and MOI. The reasons were not clear but there was no recombination in SupT1 cell lines. There was suboptimal transgene expression in primary cells. In the future, they will test AAV-Brec1 on HeLa-Smurf and Jurkat-Smurf to complement their data.  They will compare transcription levels of Brec1 mRNA in HeLa and SupT1 cells, optimize AAV transduction in SupT1/Primary T cells, and treat with compounds known to boost transgene expression in T cells. Martin shared the paper: Extracellular nanovesicles for packaging of CRISPR cas9 proteins and sgRNA to induce therapeutic exon skipping.

In the next RF3 meeting, we will brainstorm potential RF3 speakers for the HOPE Annual Meeting and go through previous actions.

Actions:

  • Kumar Lab – Send the Ott lab the mutated sequences (Sequencing clones in Jurkats, VLPs to express base editors that can mutate and fight the response) Ursula Schulze-Gahmen can test for TAR structures
  • Kumar lab – Test if you can use dead Cas9 for base editing
  • Ott and Kumar lab – Send Ulrike the constructs to test. Schedule a call to test this
  • Ulrike/Fran- send Kumar lab the constructs (dead Brec1 with fusion)
  • Niren – Combine and put a PEG coding on the PLGA, use conjugation coding, and do this at the beginning. Try more extensive pegylation
  • Kumar lab- Connect with Ursula for biochemistry for host gene targeting
  • Bring Esper onto RF3 to address their global solutions concerns
  • Michael – Share mice tissue flash freezing protocols with Priti
    • Priti can test materials sent
  • Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues
  • Ulrike meeting with Fran to look at data from a previous student for Brec off suppression
    • Ulrike-Docs inducible vectors, prepping DNA, send maps and constructs to Fran
    • Send new AV to Priti
  • Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish

 

Announcements

  • Check out the Community Jam Board to help address some of our community’s concerns
  • The HOPE Collaboratory-wide meeting is May 22nd 9am PT/12pm ET
  • The next RF3 meeting is June 5th
  • Upcoming Speakers:
    • April 24     Dr. Jon Karn
    • May 1       Dr. Jeannette Tenthorey
    • May 15     Dr. Steve Yukl
  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”