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Li-Fraumeni syndrome (LFS) is caused by a pathogenic germline variant at the TP53 locus and is associated with an increased predisposition to a variety of cancers. The neoplasms most frequently associated with LFS are sarcomas, breast cancer, brain tumors, and adrenocortical carcinomas. In this case report, we present a 43-year-old male diagnosed with an ocular adnexal sebaceous carcinoma of the right upper eyelid who was confirmed to have LFS with subsequent genetic testing. The mutational profile of both the patient's genetic screen and tumor sequencing were congruent, demonstrating the same pathogenic loss-of-function TP53 variant. This case report highlights the importance of pursuing genetic testing in patients with a history of multiple tumor types, particularly those with uncommon diagnoses. In this case, confirmation of LFS had important implications for personalized patient care, including identification of contraindicated treatment interventions and the imaging modalities necessary for vigilant follow-up screening.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (T FH ) cells, with complete restoration of BCF architecture and T FH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask T FH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy., Competing Interests: Declaration of interests The authors declare the following competing interests: H.-P.K. is or was a consultant to and has or had ownership interests in Rocket Pharmaceuticals, Homology Medicines, Vor Biopharma, and Ensoma, Inc., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Adoptive cell therapy (ACT) using T cells expressing chimeric antigen receptors (CARs) is an area of intense investigation in the treatment of malignancies and chronic viral infections. One of the limitations of ACT-based CAR therapy is the lack of in vivo persistence and maintenance of optimal cell function. Therefore, alternative strategies that increase the function and maintenance of CAR-expressing T cells are needed. In our studies using the humanized bone marrow/liver/thymus (BLT) mouse model and nonhuman primate (NHP) model of HIV infection, we evaluated two CAR-based gene therapy approaches. In the ACT approach, we used cytokine enhancement and preconditioning to generate greater persistence of anti-HIV CAR + T cells. We observed limited persistence and expansion of anti-HIV CAR T cells, which led to minimal control of the virus. In our stem cell-based approach, we modified hematopoietic stem/progenitor cells (HSPCs) with anti-HIV CAR to generate anti-HIV CAR T cells in vivo. We observed CAR-expressing T cell expansion, which led to better plasma viral load suppression. HSPC-derived CAR cells in infected NHPs showed superior trafficking and persistence in multiple tissues. Our results suggest that a stem cell-based CAR T cell approach may be superior in generating long-term persistence and functional antiviral responses against HIV infection., Competing Interests: Declaration of interests S.G.K. is a founding member of CDR3. H.-P.K. is or was a consultant to and has or had ownership interests in Rocket Pharmaceuticals, Homology Medicines, VOR Biopharma, and Ensoma., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

Hematopoietic stem cell gene therapy has been successfully used for a number of genetic diseases and is also being explored for HIV. However, toxicity of the conditioning regimens has been a major concern. Here we compared current conditioning approaches in a clinically relevant nonhuman primate model. We first customized various aspects of the therapeutic approach, including mobilization and cell collection protocols, conditioning regimens that support engraftment with minimal collateral damage, and cell manufacturing and infusing schema that reflect and build on current clinical approaches. Through a series of iterative in vivo experiments in two macaque species, we show that busulfan conditioning significantly spares lymphocytes and maintains a superior immune response to mucosal challenge with simian/human immunodeficiency virus, compared to total body irradiation and melphalan regimens. Comparative mobilization experiments demonstrate higher cell yield relative to our historical standard, primed bone marrow and engraftment of CRISPR-edited hematopoietic stem and progenitor cells (HSPCs) after busulfan conditioning. Our findings establish a detailed workflow for preclinical HSPC gene therapy studies in the nonhuman primate model, which in turn will support testing of novel conditioning regimens and more advanced HSPC gene editing techniques tailored to any disease of interest., Competing Interests: H.-P.K. has received support as the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Stephanus Family Endowed Chair for Cell and Gene Therapy and is or was a consultant to and has or had ownership interests with Rocket Pharmaceuticals, Homology Medicines, VOR Biopharma, and Ensoma Inc. H.-P.K. has also been a consultant to CSL Behring and Magenta Therapeutics. S.R. is a consultant to Forty Seven Inc. (Gilead Sciences) and Ensoma Inc., (© 2023 The Authors.)

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells' recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo -expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells' predominant CD8 + effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses., Competing Interests: Author CB is a scientific cofounder and SAB member of Catamaran Bio and Mana Therapeutics; in addition, she has patent 9,885,021 on HIV-specific T cells licensed to Mana Therapeutics. Author CB also serves on the board of directors of Cabaletta Bio and has stock ownership in Repertoire Immune Medicines and Neximmune. She also served on the DSMB for SOBI. H-PK is a scientific cofounder of Ensoma Bio. Author H-PK is a paid advisor for Ensoma Bio. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dross, Venkataraman, Patel, Huang, Bollard, Rosati, Pavlakis, Felber, Bar, Shaw, Jerome, Mullins, Kiem, Fuller and Peterson.)

Background and Aims: Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes to distinct tissues, including the liver. Vectors based on naturally occurring AAV serotypes as well as vectors using engineered capsids have shown variations in tissue tropism and level of transduction between different mouse models. Moreover, results obtained in rodents frequently lack translatability into large animal studies. In light of the increasing interest in AAV vectors for human gene therapy, an increasing number of studies are being performed in nonhuman primates. To keep animal numbers to a minimum and thus optimize the process of AAV capsid selection, we developed a multiplex barcoding approach to simultaneously evaluate the in vivo vector performance for a set of serotypes and capsid-engineered AAV vectors across multiple organs., Approach and Results: Vector biodistribution and transgene expression were assessed by quantitative PCR, quantitative reverse transcription PCR, vector DNA amplicon Illumina sequencing and vRNAseq in male and female rhesus macaques simultaneously dosed with a mixture of barcoded naturally occurring or engineered AAV vectors encoding the same transgene. As expected, our findings show animal-to-animal variation in both the biodistribution and tissue transduction pattern, which was partly influenced by each animal's distinctive serological status., Conclusions: This method offers a robust approach to AAV vector optimization that can be used to identify and validate AAV vectors for gene delivery to potentially any anatomical site or cell type., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

The bulk-boundary correspondence, which links a bulk topological property of a material to the existence of robust boundary states, is a hallmark of topological insulators. However, in crystalline topological materials the presence of boundary states in the insulating gap is not always necessary since they can be hidden in the bulk energy bands, obscured by boundary artifacts of non-topological origin, or, in the case of higher-order topology, they can be gapped altogether. Recently, exotic defects of translation symmetry called partial dislocations have been proposed to trap gapless topological modes in some materials. Here we present experimental observations of partial-dislocation-induced topological modes in 2D and 3D insulators. We particularly focus on multipole higher-order topological insulators built from circuit-based resonator arrays, since crucially they are not sensitive to full dislocation defects, and they have a sublattice structure allowing for stacking faults and partial dislocations., (© 2022. The Author(s).)

Over the past decade, numerous gene-editing platforms which alter host DNA in a highly specific and targeted fashion have been described. Two notable examples are zinc finger nucleases (ZFNs), the first gene-editing platform to be tested in clinical trials, and more recently, CRISPR/Cas9. Although CRISPR/Cas9 approaches have become arguably the most popular platform in the field, the therapeutic advantages and disadvantages of each strategy are only beginning to emerge. We have established a nonhuman primate (NHP) model that serves as a strong predictor of successful gene therapy and gene-editing approaches in humans; our recent work shows that ZFN-edited hematopoietic stem and progenitor cells (HSPCs) engraft at lower levels than CRISPR/Cas9-edited cells. Here, we investigate the mechanisms underlying this difference. We show that optimized culture conditions, including defined serum-free media, augment engraftment of gene-edited NHP HSPCs in a mouse xenograft model. Furthermore, we identify intracellular RNases as major barriers for mRNA-encoded nucleases relative to preformed enzymatically active CRISPR/Cas9 ribonucleoprotein (RNP) complexes. We conclude that CRISPR/Cas9 RNP gene editing is more stable and efficient than ZFN mRNA-based delivery and identify co-delivered RNase inhibitors as a strategy to enhance the expression of gene-editing proteins from mRNA intermediates., Competing Interests: H.P.K. has received support as the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Stephanus Family Endowed Chair for Cell and Gene Therapy, and is or was a consultant to and has or had ownership interests with Rocket Pharmaceuticals, Homology Medicines, VOR Biopharma, and Ensoma Inc. H.P.K. has also been a consultant to CSL Behring and Magenta Therapeutics. S.R. is a consultant to Forty Seven Inc. (Gilead Sciences) and Ensoma Inc., (© 2021.)

Although genome editing technologies have the potential to revolutionize the way we treat human diseases, barriers to successful clinical implementation remain. Increasingly, preclinical large animal models are being used to overcome these barriers. In particular, the immunogenicity and long-term safety of novel gene editing therapeutics must be evaluated rigorously. However, short-lived small animal models, such as mice and rats, cannot address secondary pathologies that may arise years after a gene editing treatment. Likewise, immunodeficient mouse models by definition lack the ability to quantify the host immune response to a novel transgene or gene-edited locus. Large animal models, including dogs, pigs, and non-human primates (NHPs), bear greater resemblance to human anatomy, immunology, and lifespan and can be studied over longer timescales with clinical dosing regimens that are more relevant to humans. These models allow for larger scale and repeated blood and tissue sampling, enabling greater depth of study and focus on rare cellular subsets. Here, we review current progress in the development and evaluation of novel genome editing therapies in large animal models, focusing on applications in human immunodeficiency virus 1 (HIV-1) infection, cancer, and genetic diseases including hemoglobinopathies, Duchenne muscular dystrophy (DMD), hypercholesterolemia, and inherited retinal diseases., Competing Interests: Declaration of interests H.P.K. has received support as the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Stephanus Family Endowed Chair for Cell and Gene Therapy, and is or was a consultant to and has or had ownership interests with Rocket Pharmaceuticals, Homology Medicines, VOR Biopharma and Ensoma Inc. H.P.K. has also been a consultant to CSL Behring and Magenta Therapeutics. Other authors have no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Over the past decade, chimeric antigen receptor (CAR) T cells have emerged as the prototype gene therapy for B cell leukemias. These so-called living drugs are derived from a patient's own cells, reprogrammed to recognize and destroy cancer cells, and then reintroduced into the body. The huge success of this therapy for cancer is rooted in pioneering clinical and preclinical studies, established more than three decades ago, focused on persistent HIV-1 infection. In this issue of the JCI, Bingfeng Liu et al. revisit HIV-specific CAR T cells in an important clinical study that supports broader application of this groundbreaking therapy. Although curative endpoints were not achieved, these findings lay the foundation for augmented approaches applying combinatorial technologies including antigen supplementation.

Selection of a pre-clinical non-human primate (NHP) model is essential when evaluating therapeutic vaccine and treatment strategies for HIV. SIV and SHIV-infected NHPs exhibit a range of viral burdens, pathologies, and responses to combinatorial antiretroviral therapy (cART) regimens and the choice of the NHP model for AIDS could influence outcomes in studies investigating interventions. Previously, in rhesus macaques (RMs) we showed that maintenance of mucosal Th17/Treg homeostasis during SIV infection correlated with a better virological response to cART. Here, in RMs we compared viral kinetics and dysregulation of gut homeostasis, defined by T cell subset disruption, during highly pathogenic SIVΔB670 compared to SHIV-1157ipd3N4 infection. SHIV infection resulted in lower acute viremia and less disruption to gut CD4 T-cell homeostasis. Additionally, 24/24 SHIV-infected versus 10/19 SIV-infected animals had sustained viral suppression <100 copies/mL of plasma after 5 months of cART. Significantly, the more profound viral suppression during cART in a subset of SIV and all SHIV-infected RMs corresponded with less gut immune dysregulation during acute SIV/SHIV infection, defined by maintenance of the Th17/Treg ratio. These results highlight significant differences in viral control during cART and gut dysregulation in NHP AIDS models and suggest that selection of a model may impact the evaluation of candidate therapeutic interventions for HIV treatment and cure strategies.

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro , CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo . To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Optical matter (OM) systems consist of (nano-)particle constituents in solution that can self-organize into ordered arrays that are bound by electrodynamic interactions. They also manifest non-conservative forces, and the motions of the nano-particles are overdamped; i.e., they exhibit diffusive trajectories. We propose a data-driven approach based on principal components analysis (PCA) to determine the collective modes of non-conservative overdamped systems, such as OM structures, and harmonic linear discriminant analysis (HLDA) of time trajectories to estimate the reaction coordinate for structural transitions. We demonstrate the approach via electrodynamics-Langevin dynamics simulations of six electrodynamically-bound nanoparticles in an incident laser beam. The reaction coordinate we discover is in excellent accord with a rigorous committor analysis, and the identified mechanism for structural isomerization is in very good agreement with the experimental observations. The PCA-HLDA approach to data-driven discovery of reaction coordinates can aid in understanding and eventually controlling non-conservative and overdamped systems including optical and active matter systems.

Autologous, CCR5 gene-edited hematopoietic stem and progenitor cell (HSPC) transplantation is a promising strategy for achieving HIV remission. However, only a fraction of HSPCs can be edited ex vivo to provide protection against infection. To project the thresholds of CCR5-edition necessary for HIV remission, we developed a mathematical model that recapitulates blood T cell reconstitution and plasma simian-HIV (SHIV) dynamics from SHIV-1157ipd3N4-infected pig-tailed macaques that underwent autologous transplantation with CCR5 gene editing. The model predicts that viral control can be obtained following analytical treatment interruption (ATI) when: (1) transplanted HSPCs are at least fivefold higher than residual endogenous HSPCs after total body irradiation and (2) the fraction of protected HSPCs in the transplant achieves a threshold (76-94%) sufficient to overcome transplantation-dependent loss of SHIV immunity. Under these conditions, if ATI is withheld until transplanted gene-modified cells engraft and reconstitute to a steady state, spontaneous viral control is projected to occur., Competing Interests: EC, ED, CP, DR, BM, JS No competing interests declared, HK H.-P.K has served on advisory boards for Rocket Pharmaceuticals, Homology medicines and CSL for research unrelated to this manuscript., (© 2021, Cardozo-Ojeda et al.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4 + T cells in vitro , are being explored for delivery of anti-HIV therapeutic modalities in vivo . However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.

Topological crystalline insulators (TCIs) can exhibit unusual, quantized electric phenomena such as fractional electric polarization and boundary-localized fractional charge 1-6 . This quantized fractional charge is the generic observable for identification of TCIs that lack clear spectral features 5-7 , including ones with higher-order topology 8-11 . It has been predicted that fractional charges can also manifest where crystallographic defects disrupt the lattice structure of TCIs, potentially providing a bulk probe of crystalline topology 10,12-14 . However, this capability has not yet been confirmed in experiments, given that measurements of charge distributions in TCIs have not been accessible until recently 11 . Here we experimentally demonstrate that disclination defects can robustly trap fractional charges in TCI metamaterials, and show that this trapped charge can indicate non-trivial, higher-order crystalline topology even in the absence of any spectral signatures. Furthermore, we uncover a connection between the trapped charge and the existence of topological bound states localized at these defects. We test the robustness of these topological features when the protective crystalline symmetry is broken, and find that a single robust bound state can be localized at each disclination alongside the fractional charge. Our results conclusively show that disclination defects in TCIs can strongly trap fractional charges as well as topological bound states, and demonstrate the primacy of fractional charge as a probe of crystalline topology.

Current approaches for hematopoietic stem cell gene therapy typically involve lentiviral gene transfer in tandem with a conditioning regimen to aid stem cell engraftment. Although many pseudotyped envelopes have the capacity to be immunogenic due to their viral origins, thus far immune responses against the most common envelope, vesicular stomatitis virus glycoprotein G (VSV-G), have not been reported in hematopoietic stem cell gene therapy trials. Herein, we report on two Fanconi anemia patients who underwent autologous transplantation of a lineage-depleted, gene-modified hematopoietic stem cell product without conditioning. We observed the induction of robust VSV-G-specific immunity, consistent with low/undetectable gene marking in both patients. Upon further interrogation, adaptive immune mechanisms directed against VSV-G were detected following transplantation in both patients, including increased VSV-G-specific T cell responses, anti-VSV-G immunoglobulin G (IgG), and cytotoxic responses that can specifically kill VSV-G-expressing target cell lines. A proportion of healthy controls also displayed preexisting VSV-G-specific CD4 + and CD8 + T cell responses, as well as VSV-G-specific IgG. Taken together, these data show that VSV-G-pseudotyped lentiviral vectors have the ability to elicit interfering adaptive immune responses in the context of certain hematopoietic stem cell transplantation settings., (© 2020 The Authors.)

Chimeric antigen receptor (CAR) T cells targeting CD19+ hematologic malignancies have rapidly emerged as a promising, novel therapy. In contrast, results from the few CAR T-cell studies for infectious diseases such as HIV-1 have been less convincing. These challenges are likely due to the low level of antigen present in antiretroviral therapy (ART)-suppressed patients in contrast to those with hematologic malignancies. Using our well-established nonhuman primate model of ART-suppressed HIV-1 infection, we tested strategies to overcome these limitations and challenges. We first optimized CAR T-cell production to maintain central memory subsets, consistent with current clinical paradigms. We hypothesized that additional exogenous antigen might be required in an ART-suppressed setting to aid expansion and persistence of CAR T cells. Thus, we studied 4 simian/HIV-infected, ART-suppressed rhesus macaques infused with virus-specific CD4CAR T cells, followed by supplemental infusion of cell-associated HIV-1 envelope (Env). Env boosting led to significant and unprecedented expansion of virus-specific CAR+ T cells in vivo; after ART treatment interruption, viral rebound was significantly delayed compared with controls (P = .014). In 2 animals with declining CAR T cells, rhesusized anti-programmed cell death protein 1 (PD-1) antibody was administered to reverse PD-1-dependent immune exhaustion. Immune checkpoint blockade triggered expansion of exhausted CAR T cells and concordantly lowered viral loads to undetectable levels. These results show that supplemental cell-associated antigen enables robust expansion of CAR T cells in an antigen-sparse environment. To our knowledge, this is the first study to show expansion of virus-specific CAR T cells in infected, suppressed hosts, and delay/control of viral recrudescence., (© 2020 by The American Society of Hematology.)

Spectral measurements of boundary-localized topological modes are commonly used to identify topological insulators. For high-order insulators, these modes appear at boundaries of higher codimension, such as the corners of a two-dimensional material. Unfortunately, this spectroscopic approach is only viable if the energies of the topological modes lie within the bulk bandgap, which is not required for many topological crystalline insulators. The key topological feature in these insulators is instead fractional charge density arising from filled bulk bands, but measurements of such charge distributions have not been accessible to date. We experimentally measure boundary-localized fractional charge density in rotationally symmetric two-dimensional metamaterials and find one-fourth and one-third fractionalization. We then introduce a topological indicator that allows for the unambiguous identification of higher-order topology, even without in-gap states, and we demonstrate the associated higher-order bulk-boundary correspondence., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection., Competing Interests: Declaration of Interests A.R. is an SAB member of ThermoFisher Scientific, Neogene Therapeutics, Asimov, and Syros Pharmaceuticals; a co-founder of and equity holder in Celsius Therapeutics; and an equity holder in Immunitas Therapeutics. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Cogen Therapeutics, Orche Bio, and Dahlia Biosciences. L.S.K. is on the SAB for HiFiBio; she reports research funding from Kymab Limited, Bristol Meyers Squibb, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals and consulting fees from Equillium, FortySeven, Inc, Novartis, Inc, EMD Serono, Gilead Sciences, and Takeda Pharmaceuticals. A.S. is an employee of Johnson and Johnson. N.K. is an inventor on a patent using thyroid hormone mimetics in acute lung injury that is now being considered for intervention in COVID-19 patients. J.L. is a scientific consultant for 10X Genomics, Inc. O.R.R, is a co-inventor on patent applications filed by the Broad Institute to inventions relating to single-cell genomics applications, such as in PCT/US2018/060860 and US Provisional Application No. 62/745,259. S.T. in the last three years was a consultant at Genentech, Biogen, and Roche and is a member of the SAB of Foresite Labs. M.H.W. is now an employee of Pfizer. F.J.T. reports receiving consulting fees from Roche Diagnostics GmbH and ownership interest in Cellarity, Inc. P.H. is a co-inventor on a patent using artificial intelligence and high-resolution microscopy for COVID-19 infection testing based on serology., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The transport of energy through 1-dimensional (1D) waveguiding channels can be affected by sub-wavelength disorder, resulting in undesirable localization and backscattering phenomena. However, quantized disorder-resilient transport is observable in the edge currents of 2-dimensional (2D) topological band insulators with broken time-reversal symmetry. Topological pumps are able to reduce this higher-dimensional topological insulator phenomena to lower dimensionality by utilizing a pumping parameter (either space or time) as an artificial dimension. Here we demonstrate a temporal topological pump that produces on-demand, robust transport of mechanical energy using a 1D magneto-mechanical metamaterial. We experimentally demonstrate that the system is uniquely resilient to defects occurring in both space and time. Our findings open a path towards exploration of higher-dimensional topological physics with time as a synthetic dimension.

We study nonreciprocity in spatiotemporally modulated 1D resonator chains from the perspective of equivalent 2D resonator arrays with a synthetic dimension and transverse synthetic electric and magnetic fields. The synthetic fields are respectively related to temporal and spatial modulation of the resonator chain, and we show that their combination can induce strong transmission nonreciprocity, i.e., complete isolation with only a weak perturbative modulation. This nonreciprocal effect is analogous to the Hall effect for charged particles. We experimentally implement chains of two and three spatiotemporally modulated resonators and measure over 58 dB of isolation contrast.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34 + CD90 + CD45RA - , allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Myeloid-differentiated hematopoietic stem cells (HSCs) have contributed to a number of novel treatment approaches for lysosomal storage diseases of the central nervous system (CNS), and may also be applied to patients infected with HIV. We quantified hematopoietic stem and progenitor cell (HSPC) trafficking to 20 tissues including lymph nodes, spleen, liver, gastrointestinal tract, CNS, and reproductive tissues. We observed efficient marking of multiple macrophage subsets, including CNS-associated myeloid cells, suggesting that HSPC-derived macrophages are a viable approach to target gene-modified cells to tissues. Gene-marked cells in the CNS were unique from gene-marked cells at any other physiological sites including peripheral blood. This novel finding suggests that these cells were derived from HSPCs, migrated to the brain, were compartmentalized, established myeloid progeny, and could be targeted for lifelong delivery of therapeutic molecules. Our findings have highly relevant implications for the development of novel therapies for genetic and infectious diseases of the CNS., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Recently in Nature, Gupta et al. (2019) reported that a second patient has achieved HIV-1 remission/functional cure after allogeneic hematopoietic stem cell transplantation from a donor carrying a mutation in the CCR5 coreceptor. We highlight both patients' treatments and summarize efforts to develop a broader, more patient-friendly approach., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34 + HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34 + HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

While transverse phase gradients enable studies of driven nonequilibrium phenomena in optical trapping, the behavior of electrodynamically interacting particles in a transverse phase gradient has not been explored in detail. In this Letter we study electrodynamically interacting pairs of identical nanoparticles (homodimers) in transverse phase gradients. We establish that the net driving force on homodimers is modulated by a separation-dependent interference effect for small phase gradients. By contrast, large phase gradients break the symmetry of the interaction between particles and profoundly change the electrodynamic interparticle energy landscape. Our findings are particularly important for understanding multiparticle dynamics during the self-assembly and rearrangement of optical matter.

Broadly neutralizing antibodies (bNAbs) are among the most promising strategies to achieve long-term control of HIV-1 in the absence of combination antiretroviral therapy. Passive administration of such antibodies in patients efficiently decreases HIV-1 viremia, but is limited by the serum half-life of the protein. Here, we investigated whether antibody-secreting hematopoietic cells could overcome this problem. We genetically modified human CD34 + hematopoietic stem and progenitor cells (HSPCs) to secrete bNAbs and transplanted them into immunodeficient mice. We found that the gene-modified cells engraft and stably secrete antibodies in the peripheral blood of the animals for the 9 months of the study. Antibodies were predominantly expressed by human HSPC-derived T- and B cells. Importantly, we found that secreted PGT128 was able to delay HIV-1 viremia in vivo and also prevent a decline in CD4 + cells. Gene-modified cells were maintained in bone marrow and were also detected in spleen, thymus, lymph nodes, and gut-associated lymphoid tissue. These data indicate that the bNAb secretion from HSPC-derived cells in mice is functional and can affect viral infection and CD4 + cell maintenance. This study paves the way for potential applications to other diseases requiring long-lasting protein or antibody delivery., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Competing Interests: The authors declare that they have no conflict of interest.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Purpose of Review: Combination antiretroviral therapy (ART) has enabled tremendous progress in suppressing HIV replication in infected patients. However, ART alone cannot eradicate HIV and its latent, persisting reservoirs. Novel approaches are needed to eradicate the virus or achieve functional cure in the absence of ART., Recent Findings: Adoptive T-cell therapies were initially tested in HIV-infected individuals with limited efficiency. Benefiting from new and improved methodologies, an increasing array of CAR T-cell therapies has been successfully developed in the cancer immunotherapy field, demonstrating promising new avenues that could be applied to HIV. Numerous studies have characterized various HIV-specific CAR constructs, types of cytolytic effector cells, and CAR-expressing cells' trafficking to the reservoir compartments, warranting further in-vivo efforts. Notably, the ability of CAR cells to persist and function in low-antigen environments in vivo, that is, in ART-suppressed patients, remains unclear., Summary: Despite promising results in preclinical studies, only a handful of clinical trials have been initiated worldwide. Several obstacles remain prior to successful application of HIV-specific CAR T-cell therapies in patients. In this review, we survey the current state of the field, and address paths towards realizing the goal of an efficacious HIV CAR T-cell product.

A major impediment to a more complete understanding of barrier crossing and other single-molecule processes is the inability to directly visualize the trajectories and dynamics of atoms and molecules in reactions. Rather, the kinetics are inferred from ensemble measurements or the position of a transducer ( e. g., an AFM cantilever) as a surrogate variable. Direct visualization is highly desirable. Here, we achieve the direct measurement of barrier crossing trajectories by using optical microscopy to observe position and orientation changes of pairs of Ag nanoparticles, i. e. passing events, in an optical ring trap. A two-step mechanism similar to a bimolecular exchange reaction or the Michaelis-Menten scheme is revealed by analysis that combines detailed knowledge of each trajectory, a statistically significant number of repetitions of the passing events, and the driving force dependence of the process. We find that while the total event rate increases with driving force, this increase is due to an increase in the rate of encounters. There is no drive force dependence on the rate of barrier crossing because the key motion for the process involves a random (thermal) radial fluctuation of one particle allowing the other to pass. This simple experiment can readily be extended to study more complex barrier crossing processes by replacing the spherical metal nanoparticles with anisotropic ones or by creating more intricate optical trapping potentials.

Inducing nonreciprocal wave propagation is a fundamental challenge across a wide range of physical systems in electromagnetics, optics, and acoustics. Recent efforts to create nonreciprocal devices have departed from established magneto-optic methods and instead exploited momentum-based techniques such as coherent spatiotemporal modulation of resonators and waveguides. However, to date, the nonreciprocal frequency responses that these devices can achieve have been limited, mainly to either broadband or Lorentzian-shaped transfer functions. We show that nonreciprocal coupling between waveguides and resonator networks enables the creation of devices with customizable nonreciprocal frequency responses. We create nonreciprocal coupling through the action of synthetic phonons, which emulate propagating phonons and can scatter light between guided and resonant modes that differ in both frequency and momentum. We implement nonreciprocal coupling in microstrip circuits and experimentally demonstrate both elementary nonreciprocal functions such as isolation and gyration, as well as reconfigurable, higher-order nonreciprocal filters. Our results suggest nonreciprocal coupling as a platform for a broad class of customizable nonreciprocal systems, adaptable to all wave phenomena.

Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression-significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4 + T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo , paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4 + T cells infused into HIV + patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.

The theory of electric polarization in crystals defines the dipole moment of an insulator in terms of a Berry phase (geometric phase) associated with its electronic ground state. This concept not only solves the long-standing puzzle of how to calculate dipole moments in crystals, but also explains topological band structures in insulators and superconductors, including the quantum anomalous Hall insulator and the quantum spin Hall insulator, as well as quantized adiabatic pumping processes. A recent theoretical study has extended the Berry phase framework to also account for higher electric multipole moments, revealing the existence of higher-order topological phases that have not previously been observed. Here we demonstrate experimentally a member of this predicted class of materials-a quantized quadrupole topological insulator-produced using a gigahertz-frequency reconfigurable microwave circuit. We confirm the non-trivial topological phase using spectroscopic measurements and by identifying corner states that result from the bulk topology. In addition, we test the critical prediction that these corner states are protected by the topology of the bulk, and are not due to surface artefacts, by deforming the edges of the crystal lattice from the topological to the trivial regime. Our results provide conclusive evidence of a unique form of robustness against disorder and deformation, which is characteristic of higher-order topological insulators.

[This corrects the article DOI: 10.1371/journal.ppat.1006753.].

As the HIV pandemic rapidly spread worldwide in the 1980s and 1990s, a new approach to treat cancer, genetic diseases, and infectious diseases was also emerging. Cell and gene therapy strategies are connected with human pathologies at a fundamental level, by delivering DNA and RNA molecules that could correct and/or ameliorate the underlying genetic factors of any illness. The history of HIV gene therapy is especially intriguing, in that the virus that was targeted was soon co-opted to become part of the targeting strategy. Today, HIV-based lentiviral vectors, along with many other gene delivery strategies, have been used to evaluate HIV cure approaches in cell culture, small and large animal models, and in patients. Here, we trace HIV cell and gene therapy from the earliest clinical trials, using genetically unmodified cell products from the patient or from matched donors, through current state-of-the-art strategies. These include engineering HIV-specific immunity in T-cells, gene editing approaches to render all blood cells in the body HIV-resistant, and most importantly, combination therapies that draw from both of these respective "offensive" and "defensive" approaches. It is widely agreed upon that combinatorial approaches are the most promising route to functional cure/remission of HIV infection. This chapter outlines cell and gene therapy strategies that are poised to play an essential role in eradicating HIV-infected cells in vivo.

Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.

Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based gene editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34 + cells electroporated with TALE nuclease mRNA targeting the BCL11A coding sequence. In vivo gene editing levels were low, but some BCL11A deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cell staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof-of-concept results for the selection of edited NHP CD34 + cells in culture following integration of the P140K/MGMT cassette at the BCL11A locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.

Human immunodeficiency virus (HIV) was first reported and characterized more than three decades ago. Once thought of as a death sentence, HIV infection has become a chronically manageable disease. However, it is estimated that a staggering 0.8% of the world's population is infected with HIV, with more than 1 million deaths reported in 2015 alone. Despite the development of effective anti-retroviral drugs, a permanent cure has only been documented in one patient to date. In 2007, an HIV-positive patient received a bone marrow transplant to treat his leukemia from an individual who was homozygous for a mutation in the CCR5 gene. This mutation, known as CCR5Δ32, prevents HIV replication by inhibiting the early stage of viral entry into cells, resulting in resistance to infection from the majority of HIV isolates. More than 10 years after his last dose of anti-retroviral therapy, the transplant recipient remains free of replication-competent virus. Multiple groups are now attempting to replicate this success through the use of other CCR5-negative donor cell sources. Additionally, developments in the use of lentiviral vectors and targeted nucleases have opened the doors of precision medicine and enabled new treatment methodologies to combat HIV infection through targeted ablation or down-regulation of CCR5 expression. Here, we review historical cases of CCR5-edited cell-based therapies, current clinical trials and future benefits and challenges associated with this technology., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Primary HIV-1 infection induces a virus-specific adaptive/cytolytic immune response that impacts the plasma viral load set point and the rate of progression to AIDS. Combination antiretroviral therapy (cART) suppresses plasma viremia to undetectable levels that rebound upon cART treatment interruption. Following cART withdrawal, the memory component of the virus-specific adaptive immune response may improve viral control compared to primary infection. Here, using primary infection and treatment interruption data from macaques infected with simian/human immunodeficiency virus (SHIV), we observe a lower peak viral load but an unchanged viral set point during viral rebound. The addition of an autologous stem cell transplant before cART withdrawal alters viral dynamics: we found a higher rebound set point but similar peak viral loads compared to the primary infection. Mathematical modeling of the data that accounts for fundamental immune parameters achieves excellent fit to heterogeneous viral loads. Analysis of model output suggests that the rapid memory immune response following treatment interruption does not ultimately lead to better viral containment. Transplantation decreases the durability of the adaptive immune response following cART withdrawal and viral rebound. Our model's results highlight the impact of the endogenous adaptive immune response during primary SHIV infection. Moreover, because we capture adaptive immune memory and the impact of transplantation, this model will provide insight into further studies of cure strategies inspired by the Berlin patient. IMPORTANCE HIV patients who interrupt combination antiretroviral therapy (cART) eventually experience viral rebound, the return of viral loads to pretreatment levels. However, the "Berlin patient" remained free of HIV rebound over a decade after stopping cART. His cure is attributed to leukemia treatment that included an HIV-resistant stem cell transplant. Inspired by this case, we studied the impact of stem cell transplantation in a macaque simian/HIV (SHIV) system. Using a mechanistic mathematical model, we found that while primary infection generates an adaptive immune memory response, stem cell transplantation disrupts this learned immunity. The results have implications for HIV cure regimens based on stem cell transplantation., (Copyright © 2017 American Society for Microbiology.)

The conditioning regimen used as part of the Berlin patient's hematopoietic cell transplant likely contributed to his eradication of HIV infection. We studied the impact of conditioning in simian-human immunodeficiency virus-infected (SHIV-infected) macaques suppressed by combination antiretroviral therapy (cART). The conditioning regimen resulted in a dramatic, but incomplete depletion of CD4 + and CD8 + T cells and CD20 + B cells, increased T cell activation and exhaustion, and a significant loss of SHIV-specific Abs. The disrupted T cell homeostasis and markers of microbial translocation positively correlated with an increased viral rebound after cART interruption. Quantitative viral outgrowth and Tat/rev-induced limiting dilution assays showed that the size of the latent SHIV reservoir did not correlate with viral rebound. These findings identify perturbations of the immune system as a mechanism for the failure of autologous transplantation to eradicate HIV. Thus, transplantation strategies may be improved by incorporating immune modulators to prevent disrupted homeostasis, and gene therapy to protect transplanted cells., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

The endoplasmic reticulum (ER) mediates the folding, maturation, and deployment of the secretory proteome. Proteins that fail to achieve their native conformation are retained in the ER and targeted for clearance by ER-associated degradation (ERAD), a sophisticated process that mediates the ubiquitin-dependent delivery of substrates to the 26S proteasome for proteolysis. Recent findings indicate that inhibition of long-chain acyl-CoA synthetases with triacsin C, a fatty acid analogue, impairs lipid droplet (LD) biogenesis and ERAD, suggesting a role for LDs in ERAD. However, whether LDs are involved in the ERAD process remains an outstanding question. Using chemical and genetic approaches to disrupt diacylglycerol acyltransferase (DGAT)-dependent LD biogenesis, we provide evidence that LDs are dispensable for ERAD in mammalian cells. Instead, our results suggest that triacsin C causes global alterations in the cellular lipid landscape that disrupt ER proteostasis by interfering with the glycan trimming and dislocation steps of ERAD. Prolonged triacsin C treatment activates both the IRE1 and PERK branches of the unfolded protein response and ultimately leads to IRE1-dependent cell death. These findings identify an intimate relationship between fatty acid metabolism and ER proteostasis that influences cell viability., (© 2017 To, Peterson, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well., (© 2016 by The American Society of Hematology.)

In many developing nations, cervical cancer screening is done by visual inspection with acetic acid (VIA). Monitoring and evaluation (M&E) of such screening programs is challenging. An enhanced visual assessment (EVA) system was developed to augment VIA procedures in low-resource settings. The EVA System consists of a mobile colposcope built around a smartphone, and an online image portal for storing and annotating images. A smartphone app is used to control the mobile colposcope, and upload pictures to the image portal. In this paper, a new app feature that documents clinical decisions using an integrated job aid was deployed in a cervical cancer screening camp in Kenya. Six organizations conducting VIA used the EVA System to screen 824 patients over the course of a week, and providers recorded their diagnoses and treatments in the application. Real-time aggregated statistics were broadcast on a public website. Screening organizations were able to assess the number of patients screened, alongside treatment rates, and the patients who tested positive and required treatment in real time, which allowed them to make adjustments as needed. The real-time M&E enabled by "smart" diagnostic medical devices holds promise for broader use in screening programs in low-resource settings.