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.
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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.
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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:
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Assessment of the impact of the tat degraders on tat degradation upon PROTAC cerblon knockdown in HEK293T cells
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Optimization of the following pull downs: Tat ubiquitination, tat interaction with the components of the E3 ligase complex
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Validate that the 3 tat degraders do not affect HIV integration
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Ongoing analog synthesis of TT-22856 (Bannister’s lab)
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TT-22856 was sent for pharmacokinetic analysis
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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:
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Denise – Send nonhuman primate studies/ PK experiment plan to the group soon
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Ana Leda – give DNA to Lish to look at the methylation state.
Announcements:
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If you are attending the Miami Meeting and would like to join the HOPE dinner on 12/12 Please let Robert and me know.
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The HOPE Collaboratory-wide meeting is on November 21st.
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The next RF2 Meeting is December 7th. Please come prepared with some updates.
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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.”