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RF1 Actions 7/18/22

Hello HOPEians,

Here is a quick update on the RF1 meeting from July 18th, 2022.

During the RF1 meeting, Cedric Feschotte (Cornell University) shared updates that Sabrina Leddy has started working in his lab on HERV expression in HIV elite controllers and Julius Judd who was previously working on zinc fingers is leaving his lab soon. Susana Valente (University of Florida) explained her research with DUX4, FUBP3, dCA-resistant viruses, and how p32 interacts with Tat’s basic domain.

Zichong Li (Greene Lab at Gladstone) discussed different epigenetic modifiers and the effects of new SPAs and the nucleosomal modification mechanisms for silencing HIV.  Martin Kinisu (Greene Lab) also shared his work on CRISPRoffV2 combining DNA and H3K9 methylation, the optimization of KRAB domain for HIV silencing, and exploring inducible expression systems using the piggyback technology. In his early findings, Martin found that ETRs transiently silence HIV reactivation in 30-70% of cells. Melanie Ott and Daniela Boehm (Gladstone) resubmitted their SYMD5 paper and performed the SMYD5 rescue experiment in 5A8 J-Lat cells. Their findings show that SYMD5 is highly expressed in CD4+ T cells and is a positive regulator of HIV transcription.  SYMD5 also methylates histones and Tat in vitro.

Actions for next meeting:

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

  • Chuan Li – Give MNase-Seq protocol to Zichong Li

  • 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:

  • August 15th HOPE – PAVE Guest Speaker: Dr. Persaud

  • The next standing meeting is on Sept 19 – Bharath Sreekumar will give a refresher on their data on transducing T cells.  Cedric Feschotte and Sabrina Leddy will give updates on HERV expression in HIV-1 elite controllers.

  • 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.”

RF3 Actions 6/6/22

Hello HOPEians,

In Monday’s meeting on June 6th, we heard data updates from the Chemnitz and Lange Labs. They explained the construction and functional characterization of an enzymatically dead Brec1 and the generation of different expression plasmids for brec1 and dBrec1. They also went over CD4-specific AAV2 vectors using nanobody capsid engineering. Priti gave a quick update on using CRISPR-BE (BE4) targeted to TAR to inhibit HIV; initial LTR reporter assays show reduced luciferase expression, next they will test using Ya-chi Ho’s clonal cell lines. Priti shared the paper “Efficient viral delivery of Cas9 into human safe harbor” and Nadia shared “Why drug delivery is the key to new medicines”.

There is a working group meeting on CRISPRoff efforts and relevant data on June 13th at 10am PT. Joining information is on the HOPE calendar.

Here are the updated actions from our last meeting. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • (In progress) Priti have a meeting with Ulrike 
    • Discuss CD7 nanobodies and adding RSV molecules to AAV for targeted delivery
  • Jan – Generate a Brec1 construct using an SFFV promoter for Priti

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
    • Potentially going to Yale to do initial steps on site
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The July 4th meeting is canceled for the holiday
  • The next meeting is scheduled for Aug 1st and we will host a HOPE – CRISPR for Cure Joint Meeting with Guest Speakers – Dr. Kamel Khalili and Dr. Tricia Burdo

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

RF1 Actions 5/16/22

Hello HOPEians,

Here is a quick update on the RF1 meeting from 5/16.  

During the RF1 meeting, Sonia Jablonski, a Staff Scientist in the Valente lab discussed transcriptional crosstalk between DUX4-FL and HIV-1. Martin Kinisu from the Greene lab discussed recent work on vetting CRISPR-off (7kb) as a latency promoting factor and testing variable KRAB domains. Zichong Li from the Greene lab discussed three factors that can play a role in the efforts to block & lock HIV outside of the 30 synergies identified among 10 silencing factors. Bharath Sreekumar from the Ott Lab gave an update on transducing t-cells. Thomas Premeaux shared information from the Johnathan Karn paper on The Glucocorticoid Receptor Is a Critical Regulator of HIV Latency in Human Microglial Cells. Melanie led a discussion of new ETRs including BRECiB.

 Actions for next meeting:

  • Cedric – Share data on ZNF579 with Susana
  • Roan lab – look for DUX4 expression in single-cell RNAseq tonsil datasets (+/- HIV)

Long-Term Actions:

  • Luisa Mori, Ana Leda, and Susana considering writing a review/summary of all the HIV latency models 
  • (T-TRACE) Discuss whether it would make sense to apply T-TRACE when we test block-and-lock agents in humanized mice 
  • (Systems to test spironolactone in, and which concentrations) Roan lab to collect samples to look at XPB and send to Valente Lab
  • (Aim 2) Keep the discussion open on the use of PICh or other methods

At the next meeting on June 29th, we will have a guest speaker, Dr. Mathias Lichterfeld.

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

RF3 Actions 5/3/22

Hello HOPEians,

In yesterday’s meeting, Niren Murthy gave updates about the LNP delivery systems developed in his laboratory with the goal of transfecting mRNAs in human T cells and HSCs. Stephen Yeung gave an update on the surface markers subgroup meeting where it was decided to focus on PD1 and TIGIT as markers of cells with latent HIV.

For our next meeting on June 6th, we will hear data updates from Chemnitz and Lange Labs. Here are updated actions from our meeting in May, we will discuss these at the next meeting. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • (In progress) Priti have a meeting with Ulrike 
    • Discuss CD7 nanobodies and adding RSV molecules to AAV for targeted delivery
  • Jan – Generate a BREC1 construct using an SFFV promoter for Priti
  • (In progress) Ulrike – set up a meeting with Warner to discuss good CRISPRi guides
  • All – If you have not already, please complete the HOPE Member Introduction form

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
    • Potentially going to Yale to do initial steps on site
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The next meeting is scheduled for June 6th. Chemnitz and Lange labs to present.
  • The email hope-rf3-members@gladstone.ucsf.edu can be used to reach the whole RF3 group
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form.

RF1 Actions 4/18/22

Hello HOPEians,

Here is a quick update on the RF1 meeting from 4/18.  

In today’s meeting, we discussed HIV and HERVs integration sites, with an overview from Doug, Ulrike, Matthew, and Manu. The slides will be in the RF1 Dropbox later. 

Actions for next meeting:

  • Sydney/Doug – Schedule HIV and HERVs Integration site subgroup meeting
  • Melanie – Follow up if any members of the AIDS malignancy consortium at UCSF/Gladstone. Attempt to identify HIV infected subjects on 2-deoxy-glucose treatment
  • Cedric – Share data on ZNF579 with Susana
  • Susana – Share list of ChAP-MS interactors with Cedric

Long-Term Actions:

  • Luisa Mori, Ana Leda, and Susana considering writing a review/summary of all the HIV latency models 
  • (T-TRACE) Discuss whether it would make sense to apply T-TRACE when we test block-and-lock agents in humanized mice 
  • (Systems to test spironolactone in, and which concentrations) Roan lab to collect samples to look at XPB and send to Valente Lab
  • (Aim 2) Keep the discussion open on the use of PICh or other methods

The next meeting is May 16th and RF1 leaders will follow up with a topic and agenda.

RF3 Actions 4/4/22

​​Hello HOPEians,

In yesterday’s RF3 meeting, we went over past actions items with the following updates:

  1. Phil Norris shared HIV-specific CD4+ granzyme clones with Ndhlovu and Nixon labs; they are following up with the function of these clones.
  2. Chemnitz and Lange labs working on defective BREC; constructs are made and they are testing the functionality of these constructs. They will provide an update on progress at the RF3 meeting on June 6th.
  3. Murthy lab testing lipid nanoparticle delivery in PBMCs, initial tests show low transfection efficiency, they are working on new formulations and testing in mouse models. Currently focusing on mRNA delivery instead of protein delivery of BREC due to difficulties in purification. They will provide an update on progress and discuss CAR-T cell delivery at the next meeting on May 2nd.
  4. Greene lab updated on CRISPRi work. The probability of the suppression is only good while the Cas is physically obstructing the promoter.
  5. The use of CD4 nanobodies for delivery is on pause because the molecule is not endocytosed, Priti will follow up on possibly using a CD7 nanobody. 

For our next meeting on May 2nd, we will resume our normal round table format and hear data updates from Niren. Here are updated actions from our meeting in March, we will discuss these at the next meeting. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • Priti set up a meeting with Ulrike
    • Discuss CD7 nanobodies and adding RSV molecules to AAV for targeted delivery
  • Sydney – Set up a single-cell RNA sequencing meeting
    • Invitees: Lish, Melanie, Nadia, Priti
  • Jan – Generate a BREC1 construct using an SFFV promoter for Priti
  • Melanie – send Priti a Tat expression vector using EF1a promoter (WT & defective mutant)
  • Melanie Lab – Find  a good Tat antibody for ChIP
  • Ulrike – set up a meeting with Warner to discuss good CRISPRi guides
  • All – If you have not already, please complete the HOPE Member Introduction form

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
    • Potentially going to Yale to do initial steps on site
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The next meeting is scheduled for May 2nd.
  • The email hope-rf3-members@gladstone.ucsf.edu can be used to reach the whole RF3 group
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form.

Presenters for next meeting: 

  • 5/2 – Murthy Lab and update on cell surface markers from Stephen (Ndhlovu Lab)
  • 6/6 – Update from Chemnitz and Lange Labs

RF1 Actions 3/21/22

Hello HOPEians,

Here is a quick update on the RF1 meeting from 3/21.  

In today’s meeting we got updates from:

  • Feschotte Lab: Julius presented data on ZNF579. ZNF579 interacts with TRIM22 and the PDH complex, potentially interesting for regulation of HIV latency.
  • Greene Lab: Zichong showed preliminary data that overexpressing BCL7C, KANSL2, and SIRT2 enhance HIV latency. 
  • Valente Lab: Updates on p32 regulating HIV transcription. 
  • Ott Lab: Bharath is continuing to collaborate with Ulrike to generate a telomerase immortalized primary T cell with HIV integrated into the BACH2 locus. Daniela is finalizing SMYD5 paper as well as continuing to work with the Toronto group to develop SMYD5 inhibitors (RF2).

Announcements:

  • The next meeting is April 18th – Discuss integration sites of HIV and HERVs and more lab updates

Actions for next meeting:

  • Melanie – Follow up if any members of the AIDS malignancy consortium at UCSF/Gladstone. Attempt to identify HIV infected subjects on 2-deoxy-glucose treatment
  • Cedric – Share data on ZNF579 with Susana
  • Susana – Share list of ChAP-MS interactors with Cedric
  • Subgroup meeting on ZNF579
    • Attendees: Cedric, Julius, Eric, Melanie, Susana, Warner
    • Discuss potential involvement on metabolism and interaction with components of PDH complex
  • Melanie and Susana meet to discuss potentially generating a crystal structure of p32 and Tat (Susana, Melanie, Ursula, Chuan)

Long-Term Actions:

  • Luisa Mori, Ana Leda, and Susana considering writing a review/summary of all the HIV latency models 
  • (T-TRACE) Discuss whether it would make sense to apply T-TRACE when we test block-and-lock agents in humanized mice 
  • (Systems to test spironolactone in, and which concentrations) Roan lab to collect samples to look at XPB and send to Valente Lab
  • (Aim 2) Keep the discussion open on the use of PICh or other methods