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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:

• BLE animation: This HIV Cure Strategy EXPLAINED via Animation!

• Raif’s interview with London Patient: Scientists reveal what they think of HIV Cure research!

• Raif’s interview with Patricia: HIV Cure Research & PrEP | Patricia Defechereux x Raif Derrazi

• Podcast by Instituto Saudiversidade: Spotify HIV – From Chaos to Cure Podcasts

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.”

Dear HOPEians,

Thank you for attending the first HOPE Collaboratory-Wide Meeting on March 27, 2023. Below is a summary of the meeting, actions, and announcements. 

Community: Today we heard updates and commentary from Betty Mwesigwa, Tom Villa, and Julie Ake about Uganda’s recent political legislation. “Uganda’s parliament on Tuesday passed sweeping new antigay legislation that allows death sentences for HIV-positive people engaging in repeated sexual intercourse with someone of the same gender and life imprisonment for same-sex relations.” – The Wall Street Journal 

This is devastating and alarming news and we HOPE is here to support our Ugandan colleagues. We will continue with this discussion in a separate, dedicated meeting if the community would like to express their point of view. 

RF1: Sabrina Leddy from the Feschotte Lab at Cornell presented the role of transposable elements (TEs) & KRAB zinc finger proteins (KZFPs) in HIV-1 elite controllers (EC). Some of her key takeaways are that ECs are transcriptionally heterogeneous but still distinct from healthy controls and treatment-naive viremic progressors. This heterogeneity can be resolved by grouping the ECs into four clusters, defined by their gene and TE expression (bulk RNA-seq). These cluster-specific TE signatures are corroborated by parallel bulk ATAC-seq data, showing more open chromatin at the TEs’ promoters. Potentially responsible for this upregulation, we found a number of KZFPs to be negatively correlated with the expression of cluster-specific upregulated TE families. Additionally, compared to healthy controls, ECs displayed increased chromatin accessibility at the upregulated TEs and increased expression of antiviral genes that are near these TE loci. This suggests a potential mechanism in which KZFP-mediated transcriptional silencing of specific TEs is relieved in ECs, allowing for the TEs to serve as cis-regulatory elements for adjacent genes. If true, this represents an additional dimension to HIV elite control in which immunity could be enhanced by the control of one exogenous retrovirus by another endogenous retrovirus. In the future, they plan to use scRNA-seq to reassess these dynamics in sorted infected cells, apply more locus-specific TE tools to validate our findings with STAR, incorporate new EC samples to the RNA-seq-based clustering, and validate the cis-regulatory potential of identified TE loci by KO in Jurkat cells.

RF3: Yaping Sun from the Kumar Lab at Yale presented a base editing approach to inactivate HIV-1 provirus. Yaping shared why they are using CRISPR base editing and how this works with the effective editing window. Data shows a disruption of HIV-1 TAR in the provirus with the Tat super elongation complex. The TAR editing was confirmed by sequencing. Their next steps are to look at the disruption of HIV-1 co-receptors CCR5 and CXCR4 using base editing, test base editing in primary cells, and testing in vivo. 

RF2: Ursula Schulze-Gahmen from the Ott Lab at Gladstone Institutes presented on screening for inhibitors of TAR binding to the Tat-super elongation complex. Ursula explained the role of the super elongation complex at the early active promoter and approaches to binding Tat/TAR inhibitors. She shared data from the in Vitro solution FRET Assay (HTRF) measuring TAR binding to Tat-SEC.  The assay optimizations are optimal concentration and signal stability. Positive controls found compounds as tat inhibitors. The dose-response includes a counter assay.

She referred to the paper by Ya-Chi Ho “Filgotinib suppresses HIV-1–driven gene transcription by inhibiting HIV-1 splicing and T cell activation”. Their next steps are to expand screening efforts in collaboration with NIH contractors, follow up experiments from hits from the nucleoside library, and set up a dual reporter Jurkat cell line system. 

Old Actions:

• Verdin Lab- Make a list of known targets for NF-kb cells (RNA seq) (Ideas on inactivated T cells and how disruptive this is? Hasn’t looked at other NF-kb targeting, look at IL-2 receptors)
• Lish- Send microglia cell line to Fran to test
• Lish – Follow up on the protocol for the MHRP reservoirs (leukapheresis and secretion)  Initial amendment is getting approved any day now. Protocol about reservoirs updated to include phoresis and secretions. Would these be of use to HOPE for biopsy? 

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 RF meetings: Please come prepared with some updates.
  • RF1   April 3
  • RF2   April 5
  • RF3   April 17
• 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.”

Hello HOPEians,