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Hello HOPEians,

Please find below the action items from the RF3 meeting on February 6, 2023. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar.

Updates:

Today we discussed the benefits of targeting CCR5 for mutagenesis and heard updates from the Kumar lab. The SAB suggested using a dual approach to help prevent future infections by targeting both the proviral genome and a host restriction factor. The caveat is that if only 30-40% of T cells have the CCR5 mutation, the viral rebound will undoubtedly occur prior to mutated cells taking over the cell milieu. Targeting efficiency has to be very high (perhaps >95%) and ideally be done in vivo; however, this is hard to achieve with nanoparticles. Up to a point, viral rebound is okay because you don’t change the size of the reservoir. There is an argument for going after the virus using something like laser ART in the most important tissue reservoirs where virus rebound can perhaps be staved off allowing time for repeated treatment with the gene editing approach. Laser ART is a nanoparticle-based ART approach that allows the slow release of ART at high concentrations in tissue reservoirs.

The Kumar lab is currently working on understanding the mutations induced by VLP-delivered CRISPR-base editors of HIV-1 provirus in Jurkat cells by sequencing the target. They are testing different guide RNAs and in the next couple of months hope to switch to mouse experiments. The lab is also adapting a base-editing approach for targeting CCR5 and CXCR4 for combination therapy.

Ott lab shared that Francisco transfects 30-40% TZMBL cells and then sorts cells with plasma DNA and not in Jurkats (TZM-bL cells are HELA cells with an integrated HIV-LTR luciferase reporter gene).

In the next RF3 meeting, we will hear from Fran and Ulrike about Brec1 delivery and what models they have used, and base editing

Here are some papers shared in today’s meeting:

• Functional T cells are capable of supernumerary cell division and longevity
• Methods and compositions for RNA-guided treatment of HIV infection

Actions:

• Kumar Lab – Send the Ott lab the mutated sequences obtained after base editing so Ursula Schulze-Gahmen can test for TAR structures
• Kumar lab – Test if you can use dead Brec 1 for base editing
• Ott and Kumar lab – Send Ulrike the constructs to test. Schedule a call to test this
• Ulrike/Fran- send Kumar lab the constructs (dead Brec1 with fusion)
• 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- Connect with Ursula for biochemistry for host gene targeting
• Bring Esper onto RF3 to address their global solutions concerns
• Fran – send paper
• Michael – Share mice tissue flash freezing protocols with Priti

• • Priti can test materials sent

• Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues
• Ulrike meeting with Fran to look at data from a previous student for Brec off suppression

• • Ulrike-Docs inducible vectors, prepping DNA, send maps and constructs to Fran
  • Send new AV to Priti

• Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish

Announcements:

• CRISPR for Cure – Webinar Friday, February 10th, 12-1pm PT / 3pm ET

• • Join Zoom Meeting https://temple.zoom.us/j/94549370930

• Check out the Community Jam Board to help address some of our community’s concerns
• The HOPE Collaboratory-wide meeting is on March 27.
• The next RF3 Meeting is on April 3rd. 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.”

Hello everyone,

Great working group yesterday! I will work on scheduling a follow-up Rapamycin Working Group Meeting. Here is a summary from today’s, January 17, 2023, meeting:

Eric Verdin from the Buck Institute provided background and updates on rapamycin in aging. mTOR signaling is the target of rapamycin. mTOR signaling in T cells involves metabolic cues, antigen recognition, and immunologic cues. Rapamycin can inactivate the mTORC1 complex more predominantly; however, the mTORC2 complex is inhibited with chronic rapamycin treatment. Rapamycin has been routinely used in the aging field because there is evidence that rapamycin treatment increases life span in mice  (ex: lifespan in mice can increase from 9% to 26% in almost all experiments and sometimes ranges from 30-100% increase).

Testing rapamycin in humans has not been as successful. They found that rapamycin tricks the body into thinking that there is not enough energy coming in and begins the fasting mode (even if you eat, your body thinks you are fasting). This can cause humans to lose weight and muscle. When you have high glucose levels, then insulin secretion and mTORC1 activation is triggered. There is no evidence that rapamycin works in decreasing the aging process in humans and is a drug for immunosuppression. However, there are some positive studies with primates, mice, and dogs. There is growing evidence that inactivating mTORC2 leads to side effects, whereas mTORC1 targeting may be desirable. Humans that are taking it once a week have more inhibition of mTORC1 and not mTORC2 (no side effects with 1x/week). The half-life of the drug is 36 hrs. How does it affect the lifespan of cells? In vitro replicative lifespan is generally used as a test for viability. This has not been tested in the Verdin lab, but Eric guessed that if you add rapamycin it might stop the replication of the cells. Telomere length can also be preserved.

Papers that were discussed:

Rapamycin Reduces CCR5 Density Levels on CD4 T Cells, and This Effect Results in Potentiation of Enfuvirtide (T-20) against R5 Strains of Human Immunodeficiency Virus Type 1 In Vitro.

FOXO1 promotes HIV latency by suppressing ER stress in T cells.

Regulation of stem cell function and neuronal differentiation by HERV-K via mTOR pathway

Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

This last study looks at the suppression of ART after 31 days and then administering rapamycin. Administered anti-CD3 in animals with rapamycin. Tim Henrick commented that monkey studies had really high concentrations of rapamycin (compared to his human studies). This may be because monkeys have higher levels of exposure than humans. The tissue and plasma concentrations in NHPs were way above that in humans treated long-term.

Avi Nath discusses his study: Regulation of stem cell function and neuronal differentiation by HERV-K via mTOR pathway. They showed the mTOR pathway is activated by interactions between the envelope protein of HERV-K and CD98 heavy chain. They also showed a novel interaction between mTOR and LPCAT-1 which regulates histone 4 deacetylation. In the context of human endogenous retroviruses (HERVs), there is a small number of transcription factors. HML-2 LTR can regulate the expression of host genes as promoters and enhancers. He shared some data from the paper: Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. They are interested in brain studies. If you over-express in stem cells, they will produce tumors, and the expression of neurons leads to motor neuron disease. The study shares the different effects of HML-2 expression.

Hello HOPEians,

Please find below the action items from the RF2 meeting on December 7, 2022. If you have any questions about the meeting or action items please reach out to Dr. Nadia Roan and Dr. Lish Ndhlovu. 

Updates:

Today we discussed the strengths and weaknesses from the SAB report. RF2 has done very well and the SAB did not really point out many weaknesses. One note they made was that “some candidate Tat inhibitors have EC50s in the low micromolar range and seem good candidates for optimization. However, this will require outside resources in order to pursue synthetic chemistry on several leads.” 

HOPE investigators will discuss with NIH program officers about potential strategies and resources to help with additional drug screening.  We plan to speak with Betty Poon and Gerard Lacourciere with the NIH during the Miami meeting to discuss tat-inhibitors. We could also discuss with Chris Lambros from NIAID, Avi Nath from the NIH, or Persilla Yang from Stanford about chemical biology, degraders, and protac viral targets.

Chris Lambros, PhD

National Institute of Allergy and Infectious Diseases (NIAID)

Telephone: 240-627-3093

Email: clambros@niaid.nih.gov

The next round of applications is due Jan 2023 to the Department of Health and Human Services that are internally reviewed.

HOPE investigators will reach out to Sara and Davey at the Last Gift cohort to go over potential clinical trials (combined dCA and spironolactone, Rapamycin, Brec, etc.). Clinical trials would need to be funded outside the UM1.  One potential funding source would be the ACTG:  Request for Applications- Small Clinical Trials Advancing HIV Remission and Cure (ACTG RFA). We will look at XPB degradation and Susana will discuss potential spironolactone trials to the ACTG. 

Ulrike is working with Francisco on Aim 3 using CRIPSR-based approaches. Melanie is working on dead Brec domains, but we don’t know if the effect is interfering or if this is long-lasting repression that changes the chromatin. Looking at Martin’s old work (Greene Lab), Fran is working on chromatin modifications at the LTR. We can deliver brec 1 with AV (RF3) using specific delivery methods to CD4 cells, nanobodies fused AV and higher concentrations. Priti is receiving a third batch of AAV to test on humanized mice. There is a challenge to get transgenes into T-cells lines. Other lab updates include Brazil getting their first enrollment of a patient using spironolactone.

Actions:

• Susana Valente – Coordinate with Betty Poon and Gerard Lacourciere with the NIH during the Miami meeting to discuss tat-inhibitors.

• We can connect VHL to certain binders, but how do we prove which components are binding and then develop a protac. We will coordinate with Niren to make them more permeable and maintain deficiency and solublability. 

• HOPE meetings with Last Gift

• We will look at XPB degradation and Susana will give a talk about spironolactone to the ACTG. 

• 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 January 17th. We will hear from all the RFs.  Ulrike and Fran may be presenting for RF2 (12-minute presentation, 8-minute discussion).

• The next RF2 Meeting is February 1st. 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.”

Hello HOPEians,

Happy Holidays!

During the RF1 meeting on Dec 19, 2022, we discussed some weaknesses from the SAB report and heard a lecture from Helena Reyes-Gopar (Nixon Lab) at the Instituto Nacional De Medicina Genómica about “Stellarscope quantifies the expression of transposable elements at locus resolution in single cells.” To summarize, Helena shared that they created a tool to set in motion single-cell retrotranscriptomics; they are characterizing HERV transcription in single PBMCs, and stellarscope uses a scRNA-seq alignment and your annotation to provide a matrix that includes counts for your elements (e.g HERVs).

Down the line, the Nixon Lab plans to look at more than one donor, sort T cells and monocytes, and create a small list of TEs that are expressed. Michael Corley suggested looking at methylation/chromatin data from sorted cells to orthogonally validate and also distinguish self-expressed TEs from those co-transcribed with host genes. Ulrike mentioned that they are working with bulk and mapping to HERV by taking different public datasets and found CD4 signatures. ATAC data is generally less recalcitrant to mapping because they often overlap TE and uniquely mapping flanking sequences.

One of the weaknesses that was discussed was:

“The aims of RF1 were not all totally compelling. Testing for synergy of repressive factors might be helpful but probably not groundbreaking. Looking to HERVs as models for chromatin based repression may or may not pay off – they are mostly repressed by DNA methylation in mature cells, which allows for shutoff of embryonic ZNF genes. Perhaps this is suggesting a focus on DNA methylation, though. Not sure studying tat-independent virus will be helpful.”

In the next SAB meeting, we could do better explaining the big points in the presentations and describing the physiology in more detail. We could try to understand how different HERVs located in different loci are expressed differently and look for targeted M-seq and HERV classes.

In an upcoming Scientific Working Group – HERV & HIV on January 23rd from 10-11 am PT, Cedric, Ulrike, and Zichong are working on screening zinc finger proteins for silencers and Zichong already received some good results that will be discussed further in the next meeting.

Actions for the next meeting:

• Susana Valente – Schedule meeting with Cedric to go over PITCH approach for HERVs

• 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:

• The HOPE Collaboratory-Wide Meeting is on Tuesday, January 17, 2023, 12-1 pm PT

• There is a Scientific Working Group – HERV & HIV on January 23rd from 10-11 am PT

• The next RF1 standing meeting will be in February: Date TBD and we will discuss new HERV projects

• 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,

Please find below the action items from the RF3 meeting on December 5, 2022. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar. 

Updates:

Today we discussed the SAB report including its strengths and weaknesses. It was unclear how reticuloendothelial uptake of lipid nanoparticles will be minimized. Niren explains that they need high surface coverage, so they will target liposomes with nanobodies. They will use conjugation coding at the beginning stages, to combine Peg coding. 

Another weakness was some of the work on gene excision approaches has already received approval for clinical assessment and will be restricted to HIV-infected subjects who have cancer. HOPE might consider the “Last Gift” cohort of Davey Smith at UCSD. Melanie connected Jan Chemnitz with Davey after this meeting to start potential work with “Last Gift” patients for an 8-day clinical trial. 

We will continue our collaboration with Eric Verdin for rapamycin group experiments.

The Kumar lab is working on prime editing and improving gene editing efficiencies to target more T-cells.  Additional host gene editing will be done with CCTN1 (Cyclin T1) or CCR5.  Targeting CD4+ and CD7+ cells minimizes targeting of hematopoietic stem cells.  They will look at GFP expression in humanized mice for a longer duration. Viral titers were too highly concentrated and may have precipitated.  Additional AAV2, AAV6, and AAV2/AAV6 hybrid stocks are being prepared by Ulrike’s group and will be sent to Priti’s group for testing in primary cells. 

Another concern the SAB had was the lack of attention science is giving to global solutions for cure in terms of expense and accessibility. HOPE already works with two “Global South” sites in Brazil and Uganda.  These groups could be more integrated into the RF3 program. 

Methylation is protective and continuous demethylation may cause DNA damage.  However, highly methylated DNA will be more resistant to gene editing. Michael Corley shared an article: A modular dCas9-based recruitment platform for combinatorial epigenome editing. 

Other general lab updates are: Priti is talking to the editor of Nature about her newest publication. The Kumar Lab is working with Susana Valente and Victor Garcia on a mice study. Ulrike is producing AAV and sending another batch to the Kumar Lab. She is also working on JLAT cell lines and lentiviruses with Brec1 expression. Ulrike is working on a cell line wish list for non-T-cell lines with a single copy of LTR GFP. Francisco is also interested in a reporter cell line that can be transfected with high efficiency.

The next time we meet, Fran and Michael will go over two papers they introduced today.

Actions:

• (Finished) Melanie – Connect Jan Chemnitz with Davey Smith to start work with “Last Gift” cohort

• (Finished) Melanie – Discuss with Eric Verdin about Rapamycin group experiments

• 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- Connect with Ursula for biochemistry for host gene targeting

• Bring Esper onto RF3 to address their global solutions concerns

• Fran – send paper

• Michael – Share mice tissue flash freezing protocols with Priti

  • Priti can test materials sent

• Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues

• Ulrike meeting with Fran to look at data from a previous student for Brec off suppression

  • Ulrike-Docs inducible vectors, prepping DNA, send maps and constructs to Fran

  • Send new AV to Priti

• Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish

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. 

• Check out the Community Jam Board to help address some of our community’s concerns

• The HOPE Collaboratory-wide meeting is on January 17.

• The next RF3 Meeting is February 6th. 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.”

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.”

Hello HOPEians,

Please find below the summary and action items from the RF3 meeting on October 27, 2022. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar. 

Updates:

Priti Kumar shared that her lab has started some spironolactone experiments with mice, in collaboration with the Valente lab about using pellets. In a couple of months, they will look at rebound for the silencing experiments. She has some CURE infection data with ART (only looking at nanoluciferase). 

Micahel Corely discussed that they are running new single-cell studies. They are flash-freezing tissues from mice using liquid nitrogen. He shared that the brain is the hardest to get sequencing from. Nuclei from the tissues can be isolated and it looks like it is working. 

Ulrike Lange started to test the nanobody CD4 and found transfection is successful in T cells.  Martin started experimenting with transfecting cell lines compared to primary cells and found less transfection in the primary cell system. They have been producing new batches of the nanobodies and the first batch of purified CD4-Nanobody AB6 is to be made and sent to Priti.  Priti mentioned that promoter selection will be key to determining efficacy in Rhesus macaques versus mice.  Ulrike’s group is preparing a summary of cell line models of HIV latency to test Brec1 suppression of HIV.  Ulrike will initiate MTA with Lish’s group for material transfer.  Michael proposed an experiment where GFP+/- JLAT cells were sorted prior to BREC1 transfection to test the recombinase efficacy in active and “latent” cells.  Ulrike’s group is preparing a summary of cell line models of HIV latency to test Brec1 suppression of HIV.  Ulrike will initiate MTA with Lish’s group for material transfer.  Michael proposed an experiment where GFP+/- JLAT cells were sorted prior to BREC1 transfection to test the recombinase efficacy in active and “latent” cells.

Niren Murthy updated on his LNP work.  He mentioned that negatively charged lipids can increase spleen localization.  Although this is known in non-humanized mice this effect is unknown in humanized mice.  In collaboration with the Wang lab at UC-Davis, they put IV injected neg-LNP/CRE mRNA into adult Ai9 mice (n=3).  30 minutes post-injection, spleen cells were analyzed by flow cytometry. Transfection in mice with negatively charged lipids indicates CD3+ T cells are still transfected (7.9% & 8.3% of CD3+ T cells received and expressed the transgene). Lish asked about myeloid cell transfection.  CD11C+ cells did not receive/express the transgene.  Transfection of primary human T cells with aptamer targeting LNPs is being tested for gene delivery to primary human T cells.  Preliminary data show little toxicity. Although the two formulations only showed ~1% transfection) increased doses can be tried.

Melanie Ott and Francisco Belinchon shared information about his transfection experiment and there was not a high enough efficiency with Jurkat cells. Francisco completed Brec1 (BrecOff from Ulrike) transfection in JLATs using nucleofection.  ~0.2% transfection efficiency but no effect on HIV suppression was seen.  Repeats are being done with lentiviral vectors.  Niren mentioned Brec1 is finicky. Permanent expression may be necessary and higher levels of Brec1 are required for efficacy.

-Zoom call between Ulrike and Francisco is needed next week.  

-Western blots to verify Brec1 protein expression are needed.  

-Sorting cells may be one way to delineate the effect of BrecOff in HIV suppression in JLATs.

-Francisco is still optimizing/testing the Piggyback vector and CRISPRoff system, taken over from Martin.

 -Doxycycline inducible vectors to be sent to Francisco from Ulrike’s group (maps and construct).

Actions:

• Michael – Share mouse tissue flash freezing protocols with Priti (test materials sent)

• Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues

• Ulrike meeting with Francisco to look at data from a previous student for Brec off suppression

  • Ulrike-Docs inducible vectors, prepping DNA, send maps and constructs to Francisco

  • Send new AV to Priti

• Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish

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 RF3 Meeting is December 5th. 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.”

Hello HOPEians,

During the RF1 meeting on October 17th, 2022 we discussed the new RF/Collaboratory wide format and who would submit abstracts for the HIV Persistence meeting in Miami. Attached are the Management & Operations Updates.

HIV Persistence – International Workshop Miami

Abstracts are due TODAY (by 3pm PT/ 6pm ET)

Please send me your abstracts, so we can decide who will present during the flask talks. Here is a list of individuals that are submitting abstracts (please contact me if I missed someone):

• Julie Frouard, Zichong Li, Ulrike Lange, Ursula Schulze-Gahmen, Daniela Boehm, Luisa Mori, Sonia Mediouni Jablonski, Michael Corley (2x), Alina Pang, Chuan Li, Priti Kumar

A reminder that the abstracts that you submit will likely get published if chosen. You can have max 10 authors. CROI has some rules about presenting abstracts that have already been presented:

“If study data are accepted for publication or presentation after the abstract submission to CROI, and that publication or presentation is expected to take place before CROI, the presenting author must contact the conference manager at CROIabstracts@iasusa.org to provide details as soon as the presentation is accepted for review, presentation, or publication in another venue or outlet. Please be aware that, while publication in these circumstances will not necessarily prevent presentation of the research at CROI, our strong preference is that any additional publication or presentation happens either simultaneous to or following presentation at CROI. Requests to publish or present research accepted for presentation at CROI prior to its presentation at CROI will be evaluated on a case-by-case basis. Failure to notify the conference promptly regarding plans to publish or present a CROI-accepted abstract prior to CROI may result in the removal of the study from the conference program.”

Here is some language you can use to Acknowledge HOPE & the NIH:

“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.”

New RF Structure

The RF meeting structure will change beginning in November.  For the rest of October, each RF will hold its monthly meeting.  In November, a larger group meeting will take place in lieu of the individual RF meetings.  These will alternate every month.

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.”