Blog

RF1 Actions 2/12/24

Hello HOPEians,

Here is an update on the RF1/Tat Specific meeting from February 12, 2024. If you have any questions, please reach out to Susana Valente and Doug Nixon. Meeting Recording

Actions:

  • Valente Lab – Resend XPB data to Andrew Atkins. Look at multiple time points and longitudinal data

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

Updates:

Doug led a discussion on Tat, with the main question, can Tat be targeted with active immunization to help in reservoir reduction? We reviewed the Block, Lock, Stop (BLS) program and Tat inhibition, alternative ways to “stop Tat”, and immunological intervention. The BLS approach is multipronged to drive HIV-1 from reversible latency to deep silencing and permanent transcriptional inactivation. Screening for Tat is a key component of a combination drug approach in BLS. Is there a way to stop Tat? Can we create a unique HOPE reservoir-busting immunotherapeutic? Generate anti-Tat and anti-HERV immunity?

Here are the next potential steps for HOPE:

  1. Design of Tat T cell vaccine

  2. Manufacture and testing of Tat T cell vaccine

  3. Can we identify unique HERVs expressed only as “latent cells”?

  4. Design of “latent HERV” T cell vaccine

  5. Manufacture and testing of HERV T cell vaccine

  6. Produce a HOPE Tat HERV Vaccine (HOTAHE)

There was discussion about whether the synthesis of Tat is non-toxic, T Cell responses in mouse tissue models, cytotoxic lymphocyte targetability of HIV reservoirs in the brain, and in vitro experiments to test the impact on cell types. LNP mRNA vaccines will translate to clinical trials quickly. Last Gift also has offered interventions in their cohort, could we consider Tat vaccination in that population?

Papers:

 

Announcements

  • “Cell and Gene Therapies for HIV Cure: Developing a Pipeline (P01 Clinical Trial Not Allowed).  The application due date is anticipated to be July 30, 2024

  • The next Collaboratory Wide meeting is March 25, 2024

  • Upcoming HOPE Guest Speakers:

    • Feb 26      Michael Emerman

    • Apr 22     Ivan D’Orso

    • Apr 29   Monsef Benkirane

    • May 20    Alex Marson

    • Jun 24      Mohamed Abdel-Mohsen

    • Aug 26     Judd Hultquist

  • 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 2/5/24

Hello HOPEians,

Please find below the action items from the RF3 meeting on Feb 5, 2024. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar.

Meeting Recording

New Actions:

  • Esper – Follow up with Niren to find a partner to bring LNPs to pipeline (Encapsulate mRNA)

  • Melanie – Get updates from Ursula on protein production for the Kumar Lab

  • Fran – Get an update from Reidun Twarock on sequence packaging (mathematician, packaging signals with genomes, optimizing genome sequencing)

  • Niren – Send Ott lab information and problems on Brek Protein experiments (buffer and precipitation issues)

Actions: 

  • Yaping – send instructions to Fran for transferring plasmid

  • 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 can test for TAR structures

  • Kumar lab – Test if you can you 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 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)

Updates:

Esper Kallas – global solutions focusing on the affordability and widespread availability of gene therapy approaches

Esper shared background on the Instituto Butantan with research and development infrastructure with cutting-edge technology resources. The Instituto Butantan was established 122 years ago to produce vaccines. 65% of vaccines are distributed by the Brazilian Unified Health System (SUS) and created against venomous animals, bacterial toxins, and rabies virus. The quality of chain serums is extensive and costly, involving complex processes that require live animals and many analyses. There are GLP labs (diagnosis, sequencing, immunogenicity assays) with P2 and P3 virology labs, making GMP viruses. There are non-clinical trials in line with regulatory agencies (FDA, EMA, OECD ICH, NCI, WHO). Pilot plants are egg-based platforms, prokaryotes, cell-based platforms (4 labs), monoclonal antibodies, mRNA (produce GLP material), and Zebrafish.

There are over 40 studies in the past 6 years in all regions in Brazil. Pipelines include influenza, chikungunya, NDV (COVID-19 vaccine), tetanus, diphtheria, pertussis, rabies, zika, Onco BCG, and advanced therapies (CAR-T cells, exvivo). The Insititute is developing gene therapies and investing in platforms including CAR-T cell productions for HIV and the opportunity to create a CRISPR-based treatment platform in Brazil.

Ulrike Lange – AAV9 data

Ulrike gave updates on Martin Hamann’s work from 2022 – In vivo targeting of cells by nanobody-AAV vectors. They combined CD4- and CD7-AAV6/9 hybrid capsids with single-stranded intron-containing vector genomes. The test HUSH-repression to boost transgenes expression in PBMCs (VPX/VPR, optimized intron set up). There was an exchange of CAG promoter to SFFV-presenting in vivo silencing. Packaging of adenine base-editor targeting CCR5/CXR4 host genes and large-scale AAV production for in vivo testing.

Announcements

  • SAVE the DATE– HOPE Annual Meeting will be held in NYC on Sept 30-Oct 1, 2024

  • The Progress Report process has begun.

  • 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.

    • Feb 12    RF1  Tat Specific Meeting

  • Upcoming HOPE Guest Speakers:

    • Feb 26     Michael Emerman

    • Apr 22     Ivan D’Orso

    • Apr 29     Monsef Benkirane

    • May 20    Alex Marson

    • Jun 24     Mohamed Abdel-Mohsen

    • Aug 26    Judd Hultquist

  • 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 1/22/24

Dear HOPEians,

Thank you for attending the HOPE Collaboratory-Wide Meeting on January 22, 2024. Below is a summary of the meeting, actions, and announcements.

Meeting Recording

RF2: Kallas Lab – Vivian Avelino-Silva 

“Effect of spironolactone on XPB in PLWH and PNLWH”

The Kallas lab is exploring the effects of spironolactone on XPB in PLWH and PNLWH. Vivian Avelino-Silva shared data on their global clinical investigation of candidate HIV silencing-promoting agents. They asked, is XPB expression in PLWH who are chronically exposed to SP lower when compared to PLWH who do not use SP? In their observational study, SP was mostly used for hypertension at low doses. They found that XPB levels in PLWH who were chronic SP users were not different from those observed in PLWH who were not SP users. Participants are currently undergoing leukapheresis for HIV reservoir quantification.

Next, they asked if XPB expression is reduced after exposure to SP. Their pilot project evaluated the effects of SP on XPB subunit integrity in PNLWH. They looked at relative XPB expressions before and after SP. They found no differences in XPB levels comparing pre and post measurements. The group highlighted several potential limitations including low SP dose (25mg), long frozen storage time (7 months), and intraperson variability of XPB levels. Phase 2 of the pilot study will evaluate the effects of SP use on XPB subunit integrity in PNLWH and PLWH, with several modifications in the study design and XPB measurement protocol. 

RF3: Ott Lab – Francisco Zapatero Belinchon 

“Development and delivery of next-generation epigenome editors for long-term HIV silencing”

Fran shared updates in his epigenetic regulation of HIV-1 transcription studies. The Ott Lab is testing if we could induce a programmable latent state of HIV using DNA methylation and  KRAB ZNF. In the DNA methylation, they repressed HIV-1 LTR reactivation and positively correlated with long-term aviremia PLWH. The ZNF repressed LTR by KAP1 Recruitment through the KRAB domain. Fran’s Brec1 study targets a 34-bp R region of HIV LTR with a sequence conserved in 90% HIV-1 subtypes A, B, and C and had low immunogenicity, etc. In conclusion, Fran found that BrecOFF binds HIVlTR and silence by promoter occupancy, the BrecOFF “hit and run” expression represses HIV basal transcription for 1 month, and expression does not avoid “reactivation”: in TZM-bl. He also found that ZIM2/ZIM3 KRAB domains strongly inhibit HIV LTR-driven transcription and BREcOFF expression reduced HIV reactivation in T cell line latency models. Their next steps are to conduct DNA Methylation studies, characterize BrecOFF, and optimize BrecOFF modules. 

RF1: Feschotte Lab – Sabrina Leddy

“Krab Zinc-Finger Proteins in HIV-1 infection response”

Sabrina Leddy from the Feschotte lab reminded us that their new paper is in preprint “Transposable elements may enhance antiviral resistance in HIV-1 elite controllers”.  An army of KZFP has evolved to transcriptionally silence human endogenous retroviruses (HERVs). There are nearly 400 KZFP genes in the human genome and most bind specific HERV families. There are well-studied activities in human development but barely investigated in the context of infection. They hypothesize that KZFP can impact HIV infection with direct binding to LTR or PBS sequence and place repressive marks in that way. Enabling proviral silencing.

Her indirect approach is KZFP repressing HERVs conferring regulating antiviral genes. They are characterizing the HIV-responsive HERV regulome in CD4+ T cells using a multiomic approach. They select a subset of KZFP targets with the strongest impact on HIV infection response. The TE Locus confers regulatory activity over a particular gene involved in immune response and defense. She used 25 criteria to use when selecting, for example, ZNF430, THE1B, and TRIM38.

Next, she needs to validate the role of KZFP in the CD4+ t cells hiv-responsive HERV regulome by CRISPR KO(knockout) in a CD4+ t cell line. They will start with T cells and expand to primary cells. Beyond HIV infection success in vitro and sequencing the KO cells, how else can we assess the impact of the KZFP KO on immune function? Sabrina is looking for additional metrics to measure. The Feschotte lab confirms the KZFP binding pattern in CD4+ T cells with ChIP-Exo. This study falls in line with the RF1 aims to silence HIV by targeting host and viral factors using identified host regulators of HIV proviral silencing and learning how to silence HIV from endogenous retrovirus control. 

Community: Patricia Defechereux

Community Engagement has seen significant development, setting ambitious goals for 2024. Patricia Defechereux recommends watching a captivating video featuring CAB coordinator Ebony Gordon, titled  “Songs of Our Mothers: A Singing Tree Mural.” The video showcases founding members of Healing & Uniting Every Sista, sharing their experiences as long-term survivors of HIV. The recent WAD event at Gladstone Institutes, featuring an Instagram live session with Melanie, Raif, Patricia, and Paul Edmonds Artwork, was also highlighted.

Looking ahead, an event on HIV Cure 101 in the East Bay is planned for National Black HIV Awareness Day on Feb 12th. Ifeanyi Ezeonwumelu and Alicer Andrew from the Roan Lab will be scientific speakers, and a Zoom link will be shared soon. HOPE is spearheading the planning for the Zone “CureCanvas – a Collective Vision for HIV Cure” in the Global Village at IAS 2024 in Munich, fostering collaboration across MDCs in art, science, and community. Raif will not only conduct impromptu interviews but also moderate an IAS panel on “The different flavors of gene therapy for HIV cure.” Future videos include Raif’s interview with Eric Verdin at the Buck Institute.

Additionally, Pauline and Patricia are analyzing SFAF focus group data, while Courtney Friday and the Ndhlovu lab are organizing a community event in NYC for the HOPE Annual Meeting. Patricia is organizing a CROI workshop and collaborating with Sara Gianella at the Last Gift Cohort to produce a film on “Following the Specimens.”

 

Announcements

  • SAVE the DATE– HOPE Annual Meeting will be held in NYC on Sept 30-Oct 1, 2024

  • The Progress Report process has begun.

  • 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.

    • RF3   Feb 5    ESPER – global solutions focusing on the affordability and widespread availability of gene therapy approaches, Ulrike – AAV9 data

    • RF2   Feb 7

    • RF1   Feb 12 Tat Specific Meeting

  • Upcoming HOPE Guest Speakers:

    • Feb 26     Michael Emerman

    • Apr 22     Ivan D’Orso

    • Apr 29     Monsef Benkirane

    • May 20    Alex Marson

    • Jun 24      Mohamed Abdel-Mohsen

    • Aug 26     Judd Hultquist

  • 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 12/18/23

Hello HOPEians,

Here is an update on the RF1 meeting from Dec 18, 2023. If you have any questions, please reach out to Susana Valente and Doug Nixon. Meeting Recording

Actions:

  • Valente Lab – Resend XPB data to Andrew Atkins. Look at multiple time points and longitudinal data

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

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

Updates:

Hiroyuki Matsui (Verdin Lab) – Development of metabolic intervention against HIV-1

Hiroyuki gave an update on the development of metabolic interventions against HIV-1. The susceptibility of CD4 T cells to HIV-1 infection is correlated with their activation and metabolic status. They screened a metabolite library and identified potential HIV-1 silencing metabolites. The female sex hormones and their metabolites are enriched in the silencing metabolites. The β-estradiol shows HIV-1 suppression at high concentrations but ɑ-estradiol did not, which suggests HIV-1 suppression effect is dependent on estrogen receptors. They also found that phloretin also suppressed HIV-1 reactivation, but other specific SGLT inhibitors don’t suppress HIV-1 reactivation. The Verdin Lab will continue to screen and identify potential metabolites. They will also observe dose responses to narrow down the candidates and test them in a J-lat cell lined with other types of LRAs as well as in primary DC4 T cells.

Andrew Atkins (Ndhlovu Lab) – Spironolactone in vivo Studies

RF1 aim updates:

  • RF1 aim 1: We continue to look at combinations in vitro, more updates for next call

  • RF1 aim 3: Overview of data on SP impacts in vivo

  • RF1 aim 4: Valente Lab is testing dCA treated cells, will review Luisa’s data in the next meeting

The Ndhlovu Lab is studying spironolactone effects on HIV latency in PLWH. They are specifically looking for changes in HIV DNA, XPB protein degradation, and reduction in pro-inflammatory cytokines. Andrew walked us through some of the demographics, different ART treatments, doses of either SP or amiloride, and duration of treatment. He shared results of HIV reservoir fold-change assays in participants taking SP. The CD4 count-normalized fold-change data from DNA and RNA assays coincided for all participants (linear correlation not observed). Their next steps will be to complete DNA, RNA, and protein extraction, run western blots to detect XPB degradation, and run HIV DNA and HIV RNA fold-change qPCR assays.

Paper: Estradiol and Spironolactone Plasma Pharmacokinetics Among Brazilian Transgender Women Using HIV Pre-Exposure Prophylaxis: Analysis of Potential Interactions

Announcements

  • SAVE the DATE– HOPE Annual Meeting will be held in NYC on Sept 30-Oct 1, 2024

  • The Progress Report process has begun.

  • 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   Feb 18

    • RF2   Feb 7

    • RF3   Feb 5

  • Upcoming HOPE Guest Speakers:

    • Feb 26      Michael Emerman

    • Apr 22      Ivan D’Orso

    • May 20    Alex Marson

    • Jun 24      Mohamed Abdel-Mohsen

  • 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 12/13/23

Hello HOPEians,

Please find below the action items from the RF2 meeting on Dec 13, 2023. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu. Meeting Recording

New Actions:

  • Valente Lab – will send study instructions/supplies to Esper for the pilot SP study

Actions:

  • Roan Lab – Send tonsil samples to the Valente Lab tonsil

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

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

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

Melanie Ott SMYD 5 Update
The Ott Lab has new assays with Tat substrates to screen for SMYD 5 inhibitors. They are collaborating with Brian Shoichet at UCSF and Masoud V. in Toronto to make fragment-based approaches using a published structure of the related SYMD 2 protein. They secured large amounts of purified SYMD 5 protein and are also going for a new Smyd5 structure with James Frazier at UCSF. The problem with HT screening for SMYD 5 is that regular drug libraries have few methyl transferase inhibitors. Collaborators at the Institut Pasteur in Paris are working on an epigenetic drug screen and have developed  400 compounds specific for methyltransferases. The Ott Lab just received the library and is testing the 400 compounds on SMYD 5.
Ursula is working on a Tat assay transfer to a CRO in North Carolina in collaboration with Betty Poon at NIH. They will start to screen and get results early next year.

Roan Lab Updates: Ifianyi and Kailin

Ifianyi gave updates on the primary cells in PBMCs and tonsil models. They are screening Tat degraders and ‘Block and Lock’ candidates in primary cell models of HIV infection. He shared data on the Tat inhibitors assay FACS experimental design and the infection rate vs concentration curve tested in HLAC. They will try to bring down the concentration by diluting the compounds with PBS.

Kailin showed results from the infection rate vs concentration curve tested in PBMCs. There was an increase in concentration and drop in the infection rate (ex vivo Tat PBMCs).  He shared the Tat inhibitor assay FACS experimental design and a plan to supplement with additional inhibitors. The infection rate vs concentration curve tested in HIV ‘Blok and Lock’ candidates shows infection rates are highest at 3-days. The Roan Lab tested SP and 2 drugs. When they saw inhibition, they also saw cell death, so this might not be specific to inhibiting HIV infection.

Ndhlovu Lab: Alina Pang and Andrew Atkins

Alina shared parallel data with Tat inhibitors for an old experiment based on the Tat degraders from the Valente Lab. Even with washing out, you still see reduction in reactivation. They may test this again in longer term treated patients. The DMSO control does match the dose/concentration in their myeloid cell experiments.

Andrew shared updates from the Natural History Studies cohort in vivo spironolactone studies with effects on HIV Latency in PLWH. They observed effects on XPB proteins and reservoir measurement. They found a wide range of CD4 counts and viral load. They are monitoring integration in their cell assays. HIV reservoir quantification methods include integrated HIV DNA with fold changes calculated. They found RNA signals from every sample tested. They had twice as many patients for the Amiloride treatment. The next steps are to complete the DNA/RNA/Protein extraction of the amiloride treated patients, run additional HIV DNA and XPB assays, and assess HIV-1 RNA fold-change by RT-PCR in both SP and AM.

 

Paper Shared: New findings from Emory study offer potential breakthrough in HIV cure research

We will hear from Esper and Betty in the next RF2 meeting in February.

Announcements

  • SAVE the DATE– HOPE Annual Meeting will be held in NYC on Sept 30-Oct 1, 2024

  • The Progress Report process has begun – Deadline February 1, 2024.

  • 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   Feb 18

    • RF2   Feb 7

    • RF3   Feb 5

  • Upcoming HOPE Guest Speakers:

    • Feb 26      Michael Emerman

    • Apr 22      Ivan D’Orso

    • May 20    Alex Marson

    • Jun 24      Mohamed Abdel-Mohsen

  • 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 12/4/23

Hello HOPEians,

Please find below the action items from the RF3 meeting on Dec 4, 2023. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar. 

Meeting Recording

 

New Actions:

  • Ulrike- Schedule a call with Priti to prepare a grant for AAV study

Actions: 

  • 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)

  • 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 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)

  • Priti – Test mice tissue flash-freezing protocols

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

  • Bring Esper onto RF3 to address their global solutions concerns

Updates:

Kumar Lab

Priti gave updates on her conversation with Nathan Sherer regarding individuals will CCNT1 mutations and polymorphisms in humans. They are engineering stop codons and other mutations to study the effects on immune cell types in mouse models. For delivery,  they are looking into directly introducing the mutations Iin hematopoietic stem cells. For in vivo delivery, they are also testing CD4-redirected AAVs developed by Ulrike Lange and Martin Hamann in humanized mice (in vivo). The efficiencies of CD4 nanobody AAV transduction were small in humanized mice. In the paper from the Temple group using non-targeting AAVs, the experiments show editing but not delivery. They see better excision in PBMCS but not in the tissues. They are using Laser ART which is slow-release, long-acting ART along with AAVs which they believe enables these results. Ulrike Martin’s AAV data in vitro shows lots of AAV genome presence but did not find a lot of expression. However, new data with a combination AAV6/9 capsid appears to show much higher levels of expression in PBMC after CD4 nanobody targeting.

Lange Lab

Ulrike received tissue DNA today from Priti’s lab that was extracted from huMice after AAV infection. She will test AAV content in different tissues and report backFurthermore, she is working on optimized AAV vectors for CD4 lymphocytes: First, exploring if the insertion of introns will boost expression (avoidance of HUSH silencing). This showed aeffect in primary cells. Further, changes in capsid (unblinding) are being tested for increased AAV uptake. Finally, they are trialing AAV9 serotypes that are modified to target CD4 + cells (nanobody). These can be produced at higher tighters (more than AAV6) and appear to perform better (at higher titers) than AAV6 counterpartsAt the moment they are trialing AAV9/AAV6 hybrid with nanobodies and dose escalation. They will be testing to see if it is possible to package Brec/off or Base editors on ss genomes in AAVs. Lastly, Provirex reached out to Ulrike about testing nanobodies on AAV capsids that might be possible to target hematopoietic stem cells – but needs legal pre-work by provirex.

The paper of a single AAV package is ABE- Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors

Priti shared that prime editing is too big to use for cyclin T editing. They should focus on C2i and standard base editors (need homozygous mutations). They will introduce a stop codon, loose expression on two receptors, and mutations for the HNC experiment. 

Francisco Zapatero Belinchon

Fran shared data from his brec off experiments where he observed rTA-plasmid in mice. There were many modifications and he will clone with a new promoter. He also shared data from the dox-inducible BrecOFF plasmid and the dox-inducible dBrec1 plasmid. He will test different constructs to see if BrecOff works better and clone two different Krab domains. The machine is broken so they will find a new one at Gladstone to test with.

 

Announcements

  • SAVE the DATE– HOPE Annual Meeting will be held in NYC on Sept 30-Oct 1, 2024

  • The Progress Report process will begin soon.

  • 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   Dec 18

    • RF2   Dec 13

  • Upcoming HOPE Guest Speakers:

    • Feb 26      Michael Emerman

    • Apr 22      Ivan D’Orso

    • May 20    Alex Marson

    • Jun 24      Mohamed Abdel-Mohsen

  • 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 11/27/23

Dear HOPEians,

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

Meeting recording

RF1: Ulrike Lange

Ulrike gave updates on investigating HIV-1 proviral integration site-driven proliferation dynamics in vitro. She shared data on the expansion dynamics of HIV-1 infected CD4+ T cells and how integration site can potentially drive transcription of nearby genes, including those related to cell cycle.  Ulrike shared data on the LTR cis elements as one mechanism for IS-driven expansion. The hypothesis is transcriptional activity of proviruses impacts on transcriptional activity of integration genes, which promotes cell survival/proliferation. Going forward, they are looking at in vitro selection models, workflow for integration site sequencing and clonality assessment, clonal expansion of transduced cells, qualitative evaluation of data, biological replicates and assess the effect of modulation of proviral transcriptional activity.

Shared papers:

 

Community: Patricia Defechereux

Patricia shared some upcoming events with World AIDS Day (more information to come). HOPE is hosting an Instagram live “Ask an HIV Cure Scientist” and feature some of Paul Edmond’s art display at Gladstone Institutes. Patricia is working on data analysis from the focus groups. In Brazil, Marco is leading efforts to connect with other institutions and communities to develop cure awareness activities as well as discuss identity post-cure with an upcoming stakeholder meeting. We are currently planning future events in NYC, Oakland, and Munich including the IAS Global Village. Another video project is underway, with the collaboration of the Last Gift Cohort and Sara Gianella. Additionally, updates have been made to the journal language for publication (ARHR).

 

RF2: Matt Parsons / Susana Valente

Matt Parsons gave an update on a non-human primate spironolactone dose study. They currently have three rhesus macaques getting daily oral doses of SP. After looking at PK analysis the animal study is done as well as the blood chemistries and LCMS data. Their next steps are to send shipments of NHP specimens to Valente lab, examine the western blots for XPB degradation, collect PBMC in normal EDTA and transcriptomics.

Susana shared RhM study comparison with the PK results of the  UW-Madison collaboration and AFRIMS collaboration. There was an increase in differentially expressed genes with increasing doses of SP.  Unlike in higher doses, there was no significant enrichment of inflammation-related gene pathways among differentially expressed genes with 24 mg/kg/day. Genes involved in translation are upregulated with SP treatment.

 

RF3: Melanie Ott / Priti Kumar / Niren Murthy

Melanie shared updates on their gene editing project. After attending the FDA advisory board meeting on the use of CRISPR with sickle cell anemia, she learned that CRISPR Therapeutics Announces Completion of FDA Advisory Committee Meeting for Exagamglogene Autotemcel (exa-cel) for Severe Sickle Cell Disease. The CRISPR treatment ex vivo targets the repressor of hemoglobin genes and has proven to cure sickle cell anemia. The panel was supportive of CRISPR treatment in human patients. The next steps will be cyclinT editing by Priti’s group. We will pursue two strategies –  ex vivo treatment and injection into humanized mice, and use LNPs to test both ex vivo engineering injection and direct injection of LNP base editing.

Priti shared that the prime editing approach and enzyme are large. The larger the payload is, the harder it is to package. We will incorporate mRNA and put it in hematopoietic stem cells. Niren has done two rounds of transfection and in vivo is ideal. Double-edited hematopoietic stem cells could be achievable, but there is a worry about prime editing in preclinical models.

Announcements

  • Dec 1st is World AIDS Day, join HOPE and Gladstone events!

  • SAVE the DATE– HOPE Annual Meeting will be held in NYC on Sept 30-Oct 1, 2024

  • The Progress Report process will begin soon.

  • 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   Dec 18

    • RF2   Dec 13

    • RF3   Dec 4

  • Upcoming HOPE Guest Speakers:

    • Feb 26      Michael Emerman

    • Apr 22      Ivan D’Orso

    • May 20    Alex Marson

    • Jun 24      Mohamed Abdel-Mohsen

  • 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.”

Best regards,


Sydney Norman

HOPE Collaboratory Project Coordinator
Gladstone Institutes | UCSF

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.”