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RF2 Actions 10/27/22

Hello HOPEians,

Please find below the action items from the RF2 meeting on October 27, 2022. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu. 

Updates:

Clinical Specimens

Lish gave an update on 26 patient samples from individuals on spironolactone.  Patients on eplerenone in the U.S. Military cohort were requested as a control; however, no PBMC was available.  50 controls were on amiloride.

Trevor is taking the lead for spironolactone clinical specimens.  Trevor Crowell and Brian will prepare the shipment inclusive of all controls. Collaboration between Denise/Lish/Susana -spironolactone PK in NHP samples (Matt in Thailand will send out the experimental plan to the working group).

Brazil cohort– Ana Carolina Soares mentioned that the study protocol still needs approval.  Spironolactone is not the only medication used, so enrollment is still ongoing.  No one is enrolled to date.

In vitro Experiments

Bulk RNA-seq on spironolactone-treated cells from Susana’s group may be useful in Nadia’s ex vivo primary single-cell RNA-seq study.  [~6-hour pre-treatment of spironolactone prior to infection].  Nadia Roan will continue to test PBMCs and tonsil samples. They are testing different concentrations of SP  and will block infection rates by 50% (not complete inhibition in order to leave some infected cells in spironolactone-treated condition). 

Ulrike gave an update on cell line models of HIV latency.  Generation of a cell line with a proviral reporter integrated into a region that is more heterochromatic (as reported in Elite Controllers).  CRISPR-based approaches will be used to generate these.  Alternatively, random infection and selection of clones where HIV integrates into heterochromatic regions.  Immortalization of primary cells with an hTERT plasmid – troubleshooting is underway.  Using primary T cell clones as a potential model, this needs to be optimized.

Luisa Mori is collecting data from patients treated with dCA ex vivo.  Susana discussed her group’s work on their dCA and spironolactone in combination experiments; J-Lat 10.6 cells.  The combination works better than each drug alone.  Different LRAs have been added to these conditions (8-hour LRA stimulation); GFP expression measured by flow cytometry was used as a readout.  Additionally, HIV mRNA (TAR, Gag-Pol, and Tat-Rev mRNA).  The results before Day 108 should be taken with caution due to technical issues. Focus should be placed on results past Day 108.

  • Freeze/thaw greatly affects spironolactone efficacy and should be prevented by preparing more aliquots before initial freezing.  Breakdown products of Spironolactone do not work in HIV inhibition.

  • Alina for Lish’s lab mentions that experimental time affects SP activity.  Susana’s experiment was 150 days. 

Susana also discussed Tat degrader TTX-881 plus Spironolactone combination experiments.  (all Tat degraders tested to date bind to Y26 residue on Tat).  After 12 days in culture, the combination condition inhibited HIV more than individually.  At 24 days, HIV was not as suppressed in this condition.  

Sonia Jablonski’s ongoing experiments:

  1. Assessment of the impact of the tat degraders on tat degradation upon PROTAC cerblon knockdown in HEK293T cells

  2. Optimization of the following pull downs: Tat ubiquitination, tat interaction with the components of the E3 ligase complex

  3. Validate that the 3 tat degraders do not affect HIV integration

  4. Ongoing analog synthesis of TT-22856 (Bannister’s lab)

  5. TT-22856 was sent for pharmacokinetic analysis

  6. Michael Corely / Alina Pang evaluate the 3 degraders in live cell imaging in tat 5A8 cells

Alina Pang shared some data from her dose-dependent inhibition of HIV reactivation after pre-incubation with 44863 experiments. The Tat degraders with preincubation for 5 days. She will test frozen samples for long-term effects. 

Actions:

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

  • Ana Leda – give DNA to Lish to look at the methylation state. 

Announcements:

  • If you are attending the Miami Meeting and would like to join the HOPE dinner on 12/12 Please let Robert and me know. 

  • The HOPE Collaboratory-wide meeting is on November 21st.

  • The next RF2 Meeting is December 7th. Please come prepared with some updates. 

  • 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 8/29/22

Hello HOPEians,

Please find below the action items from the RF2 meeting on August 29, 2022. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu.

Updates:

  • Lisa Henderson showed evidence that HIV transcription and translation can be observed in CSF samples from PLWH who are well-controlled on cART. The presence of these viral products is associated with neurocognitive impairment as measured by neuropsychiatric (NP) testing. They observed in vitro that targeting tat mRNA for degradation using ASOs is effective in HIV -relevant cell types (PBMC) and that knocking down tat can reduce HIV transcriptional activity and delay viral rebound; suggesting it is a viable candidate to induce ‘deep’ latency. Their next steps are to start ex-vivo experiments using patient-derived PBMCs to evaluate the effect on primary isolates. Also to start in vivo experiments in a humanized mouse model of HIV infection to evaluate the effect on tissue reservoirs and viral rebound (NIAID RAPIDD Program).

  • Susana Valente explained that they solved the issue with Tat antibodies and shared dCA and SP combination treatment in J-Lat 10.6 cells using the western blot. For the cell lines, you need to block them with milk and BSA. There is difficulty detecting Tat in PBMCs and tissue samples. Currently, they are using humanized mice and monkey tissue samples. The Brazil team might be able to study this using human sample studies, and the Roan lab can test with tonsil samples.

Actions:

  • Sara Gianella – Share ALS patient data and tissue availability with Avi Nath

    • Start a new MTA

    • Meeting with Sara Lemere, Avi Nath, Sara Weibel, and Lisa Henderson to discuss ALS & Endogenous Retrovirus reactivation in these HIV-positive participants.

  • Sara Gianella – Collaborate with Avi Nath to mine data for defective proviral sequences

  • Sara Gianella – Share morphine/opioid information on participant cohort with Lish Ndhlovu

Announcements:

  • DUE TOMORROW: Call for nominations for the HOPE 2022 Awards (Instructions attached)

  • 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 7/6/22

Hello HOPEians,

Please find below the action items from the RF2 meeting on July 6, 2022. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu.

Updates:

  • HIV Natural History Study collaboration / Trevor Crowell – New data from the exposure and timepoint definitions suggests that there are more participants than anticipated (~10) taking spironolactone and some with five or more timepoints. These longitudinal samples may include pre- and post-spironolactone administration.  Samples include PBMC but not tissues.  They are currently identifying control participants and will finalize the participant selection soon.  The MTA agreements for both Susana Valente (University of Florida) and Lish Ndhlovu (Weill Cornell) are undergoing final review and will be sent for signatures soon.

  • Sara Gianella Weibel with the Last Gift Cohort gave updates – The Last Gift (NIH P01) was recently renewed until 2027. She also shared modified autopsy procedures (Rapid autopsy → collection biobanking → processing → assays). Their new assays focus on single cell resolution (VENI and VIDI project assays).  Shared lessons learned. They found heterogeneous localization of HIV DNA within the CNS and also in the periphery.  This variability was participant-specific; however, the higher levels of HIV DNA in the spinal cord stood out and will be investigated further.  HIV RNA localization studies are ongoing and so far do not correlate with HIV DNA localization.

  • Esper Kallas & Lish Ndhlovu collaboration on tissue studies – Discussed studies for people taking spironolactone, goal is to use paired gut biopsy and peripheral blood analysis of HIV reservoir pre- and post- spironolactone treatment. First, they will look at spironolactone effects on general DNA transcription in HIV negative participants before they complete similar studies in HIV positive participants.  The IRB protocol has been submitted at USP and should have a response in approximately 30 days.  Initiation of the study can begin soon after IRB approval.

  • Susana Valente shared data from Tat degraders that were synthesized (SR-4481 & SR-44863).  High concentrations of SR-4483 was used in a preliminary murine study, but titrations will need to be done due to the high toxicity.  MTAs are ongoing with Lish Ndhlovu at Weill Cornell and Melanie Ott at UCSF.  Susana’s group has tried to validate several additional ERCC3 (XPB) antibodies, since the previously used antibody is longer available. These new antibodies do not work for WB or IP.  The Valente lab is also continuing to study the development of viruses resistant to SP. She explained the analysis of virus potentially resistant to SP used alone from the single RhM study from Maurucio Martins.  New dCA production is still 2-3 months away.  Some of this dCA is earmarked for larger NHP studies.

  • Lish shared links to:

Actions:

  • Sara Gianella – Share ALS patient data and tissue availability with Avi Nath

    • Start a new MTA

    • Meeting with Sara Lemere, Avi Nath, Sara Weibel, and Lisa Henderson to discuss ALS & Endogenous Retrovirus reactivation in these HIV positive participants.

  • Sara Gianella – Collaborate with Avi Nath to mine data for defective proviral sequences

  • Sara Gianella – Look for participants who are on spironolactone – inform Susana Valente and Nadia Roan

  • Sara Gianella – Share morphine/opioid information on participant cohort with Lish Ndhlovu

Announcements:

  • July 13 Guest Speaker: Dr. Mathias Lichterfeld

  • The next meeting is on August 3, 2022 – Lisa Henderson will present on assays for measuring defective provirsus and HIV targeting ASOs

  • If you are attending the AIDS 2022 in Montreal (July 29-August 2) please let Lish Ndhlovu and Sydney Norman know.

  • Register for the HOPE Annual Meeting

  • Call for nominations for the HOPE 2022 Awards (Instructions attached)

  • 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 6/1/22

Hello HOPEians,

Please find below the action items from the RF2 meeting on June 1st, 20222. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu

Updates:

  • Dr. Lish Ndhlovu gave updates that they are continuing to try to make dCA with chemists at Weill Cornell.
  • Dr. Brian Agan from IDCRP is joining HOPE. Several protocols have been approved to receive samples from the NHS and additional samples have been identified in RV537 of interest to HOPE.
  • Dr. Ulrike Lange has sent plasmids of different Brec1 constructs to Dr. Priti Kumar and Dr. Melanie Ott. Dr. Ulrike Lange updated about the dBrec ChIP; there was a lot of plasmid background so it was difficult to call peaks, however, it did look like there were several potential off-target sites in the genome, they will look to see if these sites contain the Brec1 recombination motif. 
  • Dr. Ana Carolina Soares gave an update that they finished writing their pilot project for spironolactone and aim to submit for IRB approval by the end of this week. They bought antibodies for western blotting for XBP so they can perform once they have samples. 

Actions:

  • Dr. Ulrike Lange – Send cloned CRISPR off construct to Dr. Melanie Ott
  • Dr. Ana Carolina Soares to send the reference for antibody for XBP to Dr. Susana Valente and Dr. Luisa Mori
  • Dr. Denise Hsu to set up a subgroup meeting to discuss a potential PK study on spironolactone in NHPs. (Valente lab, Dr. Denise Hsu and team, Dr. Mauricio Martins, and Dr. Mike Cameron)
  • Dr. Susana Valente to invite Dr. Mario Stevenson to give a talk about new myeloid cell paper
  • (In progress) Approving and obtaining MTAs from Valente Lab (Dr. Ana Leda – MTA coordinator) 
    • Adding dCA to the MTA and getting Dr. Nadia Roan an MTA
  • Dr. Susana Valente to coordinate sending DNA from dCA treated primary cells to Dr. Michael Corley to look at DNA methylation

Announcements: 

  • Dr. Melanie Ott and Dr. Ulrike Lange will be hosting a CRISPR-off Efforts and Relevant Data subgroup meeting on Monday, June 13th, 10-11am PT. If you are interested and would like to join, the information is on the HOPE Calendar.
  • Dr. Lish Ndhlovu shared the Department of Health and Human Services NIH Grant RFA
  • The next meeting is on July 6th. 

All – If you have not already, please complete the HOPE Member Introduction form

RF2 Actions 5/4/22

Hello HOPEians,

Please find below the action items from the RF2 meeting on May 4th, 2022. If you have any questions about the meeting or action items please reach out to Nadia and Lish. 

In today’s meeting we got updates from:

  • Valente Lab– Susana presented updates on her new Tat inhibitors. They are working to identify analogs with improved properties as well as continuing to study their mode of action.
  • Ndhlovu Lab – Micheal presented updates on their live-cell imaging-based reactivation system. They have added the HC69.5 microglia clone as a cell line they can test in. Additionally, presented on new enzymatic methylation sequencing with a proviral capture method which showed good coverage of the proviral sequence.

Actions:

  • Approving and obtaining MTAs from Valente Lab (Ana Leda – MTA coordinator) 
    • Adding dCA to the MTA and adding Nadia to Gladstone MTA
  • Susana to coordinate sending DNA from dCA treated primary cells to Michael Corley to look at DNA methylation

Announcements: 

  • The next meeting is on June 1st. 
  • The email hope-rf2-members@gladstone.ucsf.edu can be used to reach the whole RF2 group.
  • All – If you have not already, please complete the HOPE Member Introduction form
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form. 
  • All – Email your photo to sydney.norman@gladstone.ucsf.edu

RF2 Actions 3/2/22

Hello HOPEians,

In Wednesday’s RF2 meeting, Lisa Henderson from Avi Nath’s lab gave an overview on taking compounds from the lab to the clinic at the NIH (slides attached). Susana gave brief updates on tat inhibitor screening, and Nadia shared new results from their spironolactone experiments in primary cells.

Please find below the action items from the RF2 meeting on March 2, 2022. If you have any questions about the meeting or action items please reach out to Nadia and Lish. 

Actions:

  • All – If you have not already, please complete the HOPE Member Introduction form. Check out some of the introductions on Twitter so far!
  • All – Send Lish your photo via either email or Google Drive (*Email your photo to sydney.norman@gladstone.ucsf.edu)

Announcements: 

  • The next meeting is on April 6th.
  • The email hope-rf2-members@gladstone.ucsf.edu can be used to reach the whole RF2 group.
  • Please keep Sydney and Danielle in the loop when scheduling subgroup meetings
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form. “

The recordings from CROI discussed by Lish are available here:

RF2 Actions 2/2/22

Hello HOPEians,

Please find below the action items from the RF2 meeting on February 2, 2022. If you have any questions about the meeting or action items please reach out to Nadia and Lish. 

Actions:

  • Sydney – Schedule subgroup meeting to follow up on in vivo specimens and SP dosing (Lish, Nadia, Mauricio, Susana, Luisa, Esper, Julie)
  • All – If you have not already, please complete the HOPE Member Introduction form
  • All – Send Lish your photo via either email or Google Drive (*Email your photo to sydney.norman@gladstone.ucsf.edu)

Announcements: 

  • This invite has changed to 1 hr. Please confirm that your calendar has been updated. 
  • The next meeting is on March 2. 
  • The email hope-rf2-members@gladstone.ucsf.edu can be used to reach the whole RF2 group.
  • Please keep Sydney and Danielle in the loop when scheduling subgroup meetings
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form. 

RF2 Actions 1/5/21

Hello RF2 Rebounders,

Please find below the action items from the RF2 meeting on January 5, 2022. If you have any questions about the meeting or action items please reach out to Nadia and Lish. 

Actions:

  • Sydney – Schedule subgroup meeting to follow up on cryo-preserving humanized mouse tissue for single-cell experiments (Lish, Nadia, Priti, Susana)
  • All – Complete the RF Meeting Survey. This survey is 100% anonymous.
  • All – Send Lish your photo via either email or Google Drive (*Email your photo to sydney.norman@gladstone.ucsf.edu)
  • All – If you have not already, please complete the HOPE Member Introduction form

Announcements: 

  • The next meeting is on February 2. 
  • The email hope-rf2-members@gladstone.ucsf.edu can be used to reach the whole RF2 group.
  • Please keep Sydney and Danielle in the loop when scheduling subgroup meetings
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form. 

 

RF2 Actions 12/18/21

Hello RF2 Rebounders,

Please find below the action items from the RF2 meeting on December 8th. If you have any questions about the meeting or action items please reach out to Nadia and Lish. 

Actions:

  • Ulrike – Contact Melanie to discuss putting targeted integration constructs into immortalized primary cells
  • Nadia – Send list of compiled inhibitors and the exact compound names and distribute to the group
  • All – Send Lish your photo via either email or Google Drive (*Email your photo to sydney.norman@gladstone.ucsf.edu)
  • All – If you have not already, please complete the HOPE Member Introduction form

Announcements: 

  • The next meeting is on January 5th.
  • The email hope-rf2-members@gladstone.ucsf.edu can be used to reach the whole RF2 group.
  • Please keep Sydney and Danielle in the loop when scheduling subgroup meetings
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form.

RF2 Actions 11/3/21

Hello HOPEian Rebounders,

Here are some actions from our RF2 meeting this week (11/3). Please let me know if you have any questions or concerns.

RF2 Meeting Actions: 

Date: Nov 3, 2021

Actions:

  • Nadia – Finish compiling inhibitors and the exact compound names and distribute to the group
    • Nadia – Send information to the whole group soon
  • Nadia/Sydney – Schedule subgroup meeting to discuss compound testing and which models the compounds should be tested in (Lish, Nadia, Melanie, Warner, Susana, and Michael Corley)
    • What mechanisms are best to mix together, continue to brainstorm which inhibitors we have at our disposal, and which ones we should as a group prioritize testing.
  • All – Send Lish your photo via either email or Google Drive (*Email your photo to sydney.norman@gladstone.ucsf.edu)

Announcements: 

  • We rescheduled the RF2 meeting on Dec 1 to Wednesday Dec 8ths (1hr), Ulrike Lange will present on Aim 3
  • You can use the email hope-rf2-members@gladstone.ucsf.edu to reach the whole RF2 group
  • Please keep Sydney and Danielle in the loop when scheduling subgroup meetings