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HIV eliminated from the genomes of living animals - "Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice"
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Reported at CROI 2019:
CROI: Combination of CRISPR and LASER ART Eliminates Replication Competent Rebound in Humanized Mice
"Our study shows that treatment to suppress HIV replication and gene editing therapy, when given sequentially, can eliminate HIV from cells and organs of infected animals
"The big message of this work is that it takes both CRISPR-Cas9 and virus suppression through a method such as LASER ART, administered together, to produce a cure for HIV infection," Dr. Khalili said. "We now have a clear path to move ahead to trials in non-human primates and possibly clinical trials in human patients within the year.""
July 2, 2019
HIV eliminated from the genomes of living animals
by Temple University
https://medicalxpress.com/news/2019-07-hiv-genomes-animals.html
In a major collaborative effort, researchers at the Lewis Katz School of Medicine at Temple University and the University of Nebraska Medical Center (UNMC) have for the first time eliminated replication-competent HIV-1 DNA—the virus responsible for AIDS—from the genomes of living animals. The study, reported online July 2 in the journal Nature Communications, marks a critical step toward the development of a possible cure for human HIV infection.
"Our study shows that treatment to suppress HIV replication and gene editing therapy, when given sequentially, can eliminate HIV from cells and organs of infected animals," said Kamel Khalili, Ph.D., Laura H. Carnell Professor and Chair of the Department of Neuroscience, Director of the Center for Neurovirology, and Director of the Comprehensive NeuroAIDS Center at the Lewis Katz School of Medicine at Temple University (LKSOM). Dr. Khalili and Howard Gendelman, MD, Margaret R. Larson Professor of Infectious Diseases and Internal Medicine, Chair of the Department of Pharmacology and Experimental Neuroscience and Director of the Center for Neurodegenerative Diseases at UNMC, were senior investigators on the new study.
"This achievement could not have been possible without an extraordinary team effort that included virologists, immunologists, molecular biologists, pharmacologists, and pharmaceutical experts," Dr. Gendelman said. "Only by pooling our resources together were we able to make this groundbreaking discovery."
Current HIV treatment focuses on the use of antiretroviral therapy (ART). ART suppresses HIV replication but does not eliminate the virus from the body. Therefore, ART is not a cure for HIV, and it requires life-long use. If it is stopped, HIV rebounds, renewing replication and fueling the development of AIDS. HIV rebound is directly attributed to the ability of the virus to integrate its DNA sequence into the genomes of cells of the immune system, where it lies dormant and beyond the reach of antiretroviral drugs.
In previous work, Dr. Khalili's team used CRISPR-Cas9 technology to develop a novel gene editing and gene therapy delivery system aimed at removing HIV DNA from genomes harboring the virus. In rats and mice, they showed that the gene editing system could effectively excise large fragments of HIV DNA from infected cells, significantly impacting viral gene expression. Similar to ART, however, gene editing cannot completely eliminate HIV on its own.
For the new study, Dr. Khalili and colleagues combined their gene editing system with a recently developed therapeutic strategy known as long-acting slow-effective release (LASER) ART. LASER ART was co-developed by Dr. Gendelman and Benson Edagwa, Ph.D., Assistant Professor of Pharmacology at UNMC.
LASER ART targets viral sanctuaries and maintains HIV replication at low levels for extended periods of time, reducing the frequency of ART administration. The long-lasting medications were made possible by pharmacological changes in the chemical structure of the antiretroviral drugs. The modified drug was packaged into nanocrystals, which readily distribute to tissues where HIV is likely to be lying dormant. From there, the nanocrystals, stored within cells for weeks, slowly release the drug.
According to Dr. Khalili, "We wanted to see whether LASER ART could suppress HIV replication long enough for CRISPR-Cas9 to completely rid cells of viral DNA."
To test their idea, the researchers used mice engineered to produce human T cells susceptible to HIV infection, permitting long-term viral infection and ART-induced latency. Once infection was established, mice were treated with LASER ART and subsequently with CRISPR-Cas9. At the end of the treatment period, mice were examined for viral load. Analyses revealed complete elimination of HIV DNA in about one-third of HIV-infected mice.
"The big message of this work is that it takes both CRISPR-Cas9 and virus suppression through a method such as LASER ART, administered together, to produce a cure for HIV infection," Dr. Khalili said. "We now have a clear path to move ahead to trials in non-human primates and possibly clinical trials in human patients within the year."
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Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice
Nature Communications July 2 2019
Prasanta K. Dash1,4, Rafal Kaminski2,4, Ramona Bella2,4, Hang Su1, Saumi Mathews1, Taha M. Ahooyi2,
Chen Chen2, Pietro Mancuso2, Rahsan Sariyer2, Pasquale Ferrante2, Martina Donadoni2, Jake A. Robinson2, Brady Sillman1, Zhiyi Lin1, James R. Hilaire1, Mary Banoub1, Monalisha Elango1, Nagsen Gautam3, R. Lee Mosley1, Larisa Y. Poluektova1, JoEllyn McMillan1, Aditya N. Bade1, Santhi Gorantla1, Ilker K. Sariyer2, Tricia H. Burdo2, Won-Bin Young2, Shohreh Amini2, Jennifer Gordon2, Jeffrey M. Jacobson2, Benson Edagwa1, Kamel Khalili2 & Howard E. Gendelman1
Abstract
Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.
Discussion
While ART has transformed HIV-1 infection into a chronic treatable disease, virus persists in tissues that include the gut, lymph nodes, brain, spleen amongst other sites. The inability of ART to eliminate virus in these tissue sanctuaries remains the major obstacle towards a disease cure. Such a limitation is linked, in large measure, to continuous long-term infections in CD4 + memory T cells and less frequently in mononuclear phagocytes despite both directed host antiviral immunity and ART effectiveness. Thus, one may predict that, any or all steps towards HIV elimination must include precise targeted ART delivery, maintenance of vigorous immune control, effective blockade of viral growth and immune-based elimination of pools of infected cells or genome integrated proviral DNA. Even under these conditions, the presence of replication competent virus that allows low-levels of viral production and viral latency underscores employment of strategies that eliminate virus that is integrated but latent. Because of notable graft versus host disease in several humanized animal models, examinations for time periods measured in months are limited. In order to overcome the challenge of sustained human grafts in mice, we adopted NSG-humanized mice transplanted at birth with HSC. Both human myeloid and lymphoid lineages were successfully reconstituted in these mice and support the evaluations of HIV-1 persistence, treatment, and immune functions17,30,31,32,33,34,35,36,37,38. The sustained human grafts as confirmed by flow cytometry were viable and functional for more than 6 months, which provided a platform that allowed treatment interventions for prolonged time periods and a clear ability during ART to best establish a continuous latent HIV-1 reservoir in peripheral tissues and the brain and the noted immunological responses to the viral infection12,17,29,36,37,39. These previously published data support the successful use of humanized mice in studies of HIV/AIDS pathogenesis, therapeutics40,41,42, and treatment12,13,14,16,18,29,43,44. These studies, taken together, clearly provide a rationale for the scientific approaches taken in the current report12,13,29,43,45,46,47.
Therefore, our approaches towards evaluating viral cures have included the demonstrated ability of the drugs to reach sites of latent infection and to do so at significant levels18,37,39,43,44. Notably, the use of molecular tools can permanently eliminate the viral genome and preclude reactivation20,21,24,48. Thus, we suggest that the current successful outcome in achieving this goal in more than 30% of the infected experimental animals reflects the combinatorial use of a suitable animal model, control of viral set points, reach to the viral reservoirs, delivery and intracellular drug penetration of potent LASER ART, and the widespread employment of CRISPR-Cas9 gene editing. The latter enabled high efficiency excision of large fragments of the viral genome from anatomically privileged tissues. Results support the idea that maximal viral restriction must be first established prior to excision to achieve optimal viral editing by CRISPR-Cas9.
Current HIV-1 treatment patterns are defined by daily dosing of a combination of either two nucleoside reverse transcriptase inhibitors (NRTIs) and one integrase strand transfer inhibitor (INSTI), or two NRTIs and one nonnucleoside reverse transcriptase inhibitor. Rebound that follows affects both the number and function of CD4 + T cells leading to virus-associated co-morbid conditions. LASER ART was developed in an attempt to eliminate these limitations and was shown effective in establishing drug depots in macrophages with sustained antiretroviral activities and reductions in HIV-1 proviral load beyond ART alone15,38,47,49,50,51,52,53,54,55. The success in these prior studies led to the use of LASER ART in the current report in order to maximize ART ingress to cell and tissue sites of viral replication enabling the drugs to reach these sites at high concentrations for sustained time periods. The maintenance of slow drug release for times measured in weeks or longer provided optimal settings for viral excision17,39,47. ART particles coated with poloxamers enabled lipophilic hydrophobic prodrug crystals to readily cross cell and tissue barriers, aiding precision drug release to viral sanctuary sites12,13,14,37,39,46. These claims are reinforced by our prior studies demonstrating up to a 10-fold increase in viral restriction at two independent multiplicities of infection in CD4 + T cell lines with LASER ART when compared to conventional native drugs12,13. The advantages of LASER ART over native ART include rapid entry across cell membranes of both CD4 + T cells and macrophages (due to drug lipophilicity); accelerated antiretroviral drug entry into viral reservoir sites (including the brain, gut, lymph nodes, liver, bone marrow and spleen); increased intracellular drug delivery; and stable plasma concentrations observed over weeks to months. The ART were selected in order to produce sustained plasma concentrations 4X the protein-adjusted 90% inhibitory concentration. Notably, a single parenteral dose of NMDTG at 45 mg DTG equivalents/kg to mice provided plasma DTG concentration of 88 ng/ml at 56 days32. Liver, spleen and lymph node DTG concentrations were 8.0, 31.2 and 17.6 ng/g, respectively at 56 days following single treatment. At 14 days after NMABC and NM3TC given at 50 mg ABC or 3TC equivalents/kg to mice, ABC and 3TC plasma concentrations were 21 and < 7 ng/ml, respectively12,13,14. In summary, there was little to no residual ART in plasma or tissue at the time of animal sacrifice reflecting the robust viral rebound found in all infected mice treated with LASER ART alone. Further, significant efforts were made by us to demonstrate that one month after LASER ART was discontinued, viral rebound was detectable. All of this highlights the rationale for use of LASER ART over native ART. Most importantly, our research12,13,14 demonstrated that ART levels in plasma were undetectable during the period of measured viral rebound.
For elimination of proviral DNA, we chose the CRISPR-Cas9 gene editing platform and created a multiplex of gRNAs that caused cleavage of the viral genome at the highly conserved regions within the LTRs and the Gag gene. This strategy allowed for the removal of the large intervening DNA fragments across the viral genome and mitigated any chance for the emergence of virus escape mutants20,23. In support of this notion, results from cell culture and animal adoptive infection studies showed the absence of replication competent HIV-1 in the spleen and bone marrow of animals with no rebound that could be attributed to virus escape. Our choice for the use of AAV9 comes from earlier studies demonstrating the broad range tissue distribution of CRISPR-Cas9 in a mouse model20. Accordingly, the results in our current study verified the bioavailability of our gene editing molecule in various organs of the NSG humanized mice. No off-target effects were detected in in vivo deep sequencing and bioinformatics analysis that may be caused by the CRISPR-Cas9 editing strategy. Nevertheless, as expected naturally occurring cellular DNA variation was found in both untreated cells as well as in CRISPR-Cas9-treated cells. Examination of several potential target cellular genes performed on clonal cells expressing CRISPR-Cas9 by gene amplification and direct sequencing showed no mutations that may be caused by the presence of CRISPR-Cas9 in the cells.
Results from ddPCR showed 60% to 80% efficiency of viral DNA excision by CRISPR-Cas9. Of note, this approach quantified dual cleavage events that removed the DNA fragment spanning 5'LTR to 3'LTR, 5'LTR to gag, and gag to 3'LTR of the proviral genome. However, the occurrence of single site editing events that would permanently interrupt the viral DNA and potentially inactivate viral replication by introducing small InDel mutations at the cleavage sites are not included in this estimate19. Therefore, viral activation and rebound may not be observed under the conditions whereby excision efficiency is less than 100%. In recent studies, we demonstrated that inclusion of quadruplex of gRNAs for targeting Gag, Pol and two separate sites within the LTRs may yield slightly higher efficiency of viral DNA excision24. It is important to note that in those studies we employed a different mouse model, distinct AAV delivery system, and more importantly, different timelines for HIV-1 infection and viral DNA harvesting for conventional semi-quantitative PCR assays. In recent studies, we combined bioimaging, antiretroviral PK and sensitive tissue biodistribution studies to facilitate ART delivery into cell and tissue viral reservoirs in both humanized mice and non-human primates. These combined diagnostic and therapeutic modalities, coined theranostics, are being developed to facilitate effective HIV-1 elimination strategies in an infected human host56.
In conclusion, we employed a broad range of highly sensitive tests to evaluate HIV-1 elimination by LASER ART and AAV9-delivered CRISPR-Cas9 treatments. These included viral gene amplification, flow cytometry, adoptive viral transfers, on target and off target assays, and measures of viral rebound to demonstrate that combination therapies can safely lead to the elimination of HIV-1 infection. Results demonstrated that eradication of replication-competent HIV-1 present in infectious cell and tissue sites of infected animals can be achieved. Although reappearance of viremia in humans can be delayed6, rebound occurs on average 2 to 4 weeks after ART interruption6,57,58 and 5 to 11 days in humanized mice59. Despite the vigorous treatments offered, there was no evidence of outward untoward effects of any therapies (supplementary fig. 19)12 including the persistence of human adult lymphocytes in mouse plasma and tissue (Fig. 4). As such, these proof-of-concept results offer readily defined and realistic pathways toward strategies for HIV-1 elimination. Future studies are designed to improve delivery of agents to viral reservoirs and specifically eliminate residual latent viral infections. This is a first important step towards a longer journey for viral eradication.
Introduction
According to UNAIDS, it is estimated that more than 36.7 million people worldwide are infected with the human immunodeficiency virus type one (HIV-1) and >5000 individuals worldwide are newly infected each day. In the clinic, antiretroviral therapy (ART) restricts viral infection by stalling various steps of the viral life cycle. However, ART fails to eliminate integrated copies of HIV-1 proviral DNA from the host genome1,2. As such, virus persists in a latent state within infectious reservoirs; and ART cessation readily leads to viral reactivation and disease progression to acquired immunodeficiency syndrome (AIDS)3. Thus, a major issue for any HIV-1 curative strategy is the means to eliminate either integrated proviral DNA or the cells that harbor virus without collateral cytotoxic reactions. However, elimination of HIV-1 infection in its infected human host is documented only in two individuals4,5. There are several reasons why success has not yet been realized. This includes inadequate therapeutic access to viral reservoirs, rapid spread of infection by continuous sources of virus and susceptible cells and a failure to eliminate residual latent integrated proviral DNA. All single or combination therapeutic approaches preclude HIV-1 cure as viral rebound universally follows ART cessation6,7,8,9,10. Yet, another obstacle towards elimination of infection is that viral latency is established after infection onset and precedes peak viremia. This underscores that early intervention with potent antiretroviral medicines may help to further reduce the size of the reservoir and ultimately facilitate viral elimination11. Therefore, multimodal robust pharmaceutic strategies are needed for complete elimination of HIV-1 if no viral resurgence after cessation of ART is to be achieved. To address this need and design a suitable therapeutic strategy, our laboratories produce highly hydrophobic lipophilic viral reservoir penetrating antiretroviral prodrugs coined as long-acting slow-effective release ART (LASER ART). LASER ART properties are defined by slow drug dissolution, enhanced lipophilicity, improved bioavailability and limited off-target toxicities, which directly affect the frequency of ART administration from daily to weeks. These reduce disease co-morbidity in small animals and maintains effective antiretroviral drug concentrations in blood and tissue viral reservoirs from days to weeks12,13,14,15,16. Macrophages enable uptake of significant amounts of intracellular antiretroviral drug crystals and tightly control ongoing viral replication by the cells' slow drug release and transfer to adjacent CD4 + T cells during cell-to-cell contact or through direct drug uptake13,14,16,17,18.
However, LASER ART alone cannot rid the infected host of latent HIV-1 no matter how successful the drugs may prove to be at restricting viral infection. Thus, in parallel, we develop CRISPR-Cas9 based gene editing technology using AAV9 delivery that specifically and efficiently excises fragments of integrated HIV-1 proviral DNA from the host genome19,20,21,22,23,24. We realize that CRISPR-Cas9-based technologies could be most effective in the setting of maximal viral restriction and substantive reductions in the absolute proviral DNA load. Thus, the two approaches are combined to examine whether LASER ART and CRISPR-Cas9 treatments could provide combinatorial benefit for viral elimination. Here we demonstrate elimination of replication competent HIV-1 in an experimental model of human infectious disease. Viral clearance is achieved from HIV-1 infected spleen and lymphoid tissues as well as a broad range of solid organs from documented prior infected humanized mice treated with LASER ART and AAV9-CRISPR-Cas9. This is confirmed in those mice using ultrasensitive HIV-1 nucleic acid detection methods by the absence of post-treatment viral rebound; and by the inability to transfer virus from those infected and dual-treated mice to replicate uninfected untreated mice. We conclude that viral elimination by a combination of LASER ART and gene editing strategy is possible.
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