RESUMEN
With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.
Asunto(s)
COVID-19 , Inhibidores de Fusión de VIH , Animales , Ratones , Humanos , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Ratones Transgénicos , Péptidos/farmacologíaRESUMEN
The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein's function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.
Asunto(s)
Vectores Genéticos/fisiología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Neutralizantes/inmunología , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Lentivirus/genética , Mutación , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Carga Viral/genéticaRESUMEN
Cell therapies based on reprogrammed adaptive immune cells have great potential as "living drugs." As first demonstrated clinically for engineered chimeric antigen receptor (CAR) T cells, the ability of such cells to undergo clonal expansion in response to an antigen promotes both self-renewal and self-regulation in vivo. B cells also have the potential to be developed as immune cell therapies, but engineering their specificity and functionality is more challenging than for T cells. In part, this is due to the complexity of the immunoglobulin (Ig) locus, as well as the requirement for regulated expression of both cell surface B cell receptor and secreted antibody isoforms, in order to fully recapitulate the features of natural antibody production. Recent advances in genome editing are now allowing reprogramming of B cells by site-specific engineering of the Ig locus with preformed antibodies. In this review, we discuss the potential of engineered B cells as a cell therapy, the challenges involved in editing the Ig locus and the advances that are making this possible, and envision future directions for this emerging field of immune cell engineering.
Asunto(s)
Linfocitos B/metabolismo , Sistemas CRISPR-Cas , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Edición Génica , Terapia Genética/métodos , Inmunoterapia/métodos , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Linfocitos B/inmunología , Ingeniería Celular , Reprogramación Celular/genética , Reprogramación Celular/inmunología , Regulación de la Expresión Génica , Ingeniería Genética , Humanos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Homology-directed repair (HDR) of a DNA break allows copying of genetic material from an exogenous DNA template and is frequently exploited in CRISPR-Cas9 genome editing. However, HDR is in competition with other DNA repair pathways, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), and the efficiency of HDR outcomes is not predictable. Consequently, to optimize HDR editing, panels of CRISPR-Cas9 guide RNAs (gRNAs) and matched homology templates must be evaluated. We report here that CRISPR-Cas9 indel signatures can instead be used to identify gRNAs that maximize HDR outcomes. Specifically, we show that the frequency of deletions resulting from MMEJ repair, characterized as deletions greater than or equal to 3 bp, better predicts HDR frequency than consideration of total indel frequency. We further demonstrate that tools that predict gRNA indel signatures can be repurposed to identify gRNAs to promote HDR. Finally, by comparing indels generated by S. aureus and S. pyogenes Cas9 targeted to the same site, we add to the growing body of data that the targeted DNA sequence is a major factor governing genome editing outcomes.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Reparación del ADN por Unión de Extremidades , Edición Génica , Mutación INDEL , ARN Guía de Kinetoplastida/genética , Reparación del ADN por Recombinación , Proteína 9 Asociada a CRISPR/genética , Roturas del ADN de Doble Cadena , Células HEK293 , Humanos , Células K562RESUMEN
Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 µg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 µg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 µg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.
Asunto(s)
Antígenos CD4/inmunología , Dependovirus/genética , Inmunoglobulinas/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Internalización del Virus , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Antagonistas de los Receptores CCR5/inmunología , Antígenos CD4/genética , Femenino , Terapia Genética , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , VIH-2/inmunología , Inmunoglobulinas/genética , Macaca mulatta , Masculino , Pruebas de Neutralización , Receptores CCR5/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virologíaRESUMEN
Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.
Asunto(s)
VIH-1/inmunología , Latencia del Virus/inmunología , Latencia del Virus/fisiología , Animales , Antirretrovirales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Modelos Animales de Enfermedad , Infecciones por VIH/virología , Seropositividad para VIH/tratamiento farmacológico , VIH-1/patogenicidad , Humanos , Ratones , Receptor de Muerte Celular Programada 1/inmunología , Receptores Inmunológicos/inmunología , Nucleasas de los Efectores Tipo Activadores de la Transcripción/inmunología , Activación Viral , Replicación ViralRESUMEN
Adeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem and progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 and AAVS1 loci in human HSPCs and cell lines. As a control, we used AAV6, which effectively edits HSPCs but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc-finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results, therefore, do not support that clade F AAVs can perform high-efficiency genome editing in the absence of a DSB.
Asunto(s)
Roturas del ADN de Doble Cadena , Dependovirus/fisiología , Edición Génica/métodos , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/citología , Células Cultivadas , Dependovirus/clasificación , Dependovirus/genética , Marcación de Gen , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Células Madre Hematopoyéticas/metabolismo , Recombinación Homóloga , Humanos , Células K562 , Receptores CCR5/genética , Ensamble de VirusRESUMEN
Most studies of HIV latency focus on the peripheral population of resting memory T cells, but the brain also contains a distinct reservoir of HIV-infected cells in microglia, perivascular macrophages, and astrocytes. Studying HIV in the brain has been challenging, since live cells are difficult to recover from autopsy samples and primate models of SIV infection utilize viruses that are more myeloid-tropic than HIV due to the expression of Vpx. Development of a realistic small animal model would greatly advance studies of this important reservoir and permit definitive studies of HIV latency. When radiation or busulfan-conditioned, immune-deficient NSG mice are transplanted with human hematopoietic stem cells, human cells from the bone marrow enter the brain and differentiate to express microglia-specific markers. After infection with replication competent HIV, virus was detected in these bone marrow-derived human microglia. Studies of HIV latency in this model would be greatly enhanced by the development of compounds that can selectively reverse HIV latency in microglial cells. Our studies have identified members of the CoREST repression complex as key regulators of HIV latency in microglia in both rat and human microglial cell lines. The monoamine oxidase (MAO) and potential CoREST inhibitor, phenelzine, which is brain penetrant, was able to stimulate HIV production in human microglial cell lines and human glial cells recovered from the brains of HIV-infected humanized mice. The humanized mice we have developed therefore show great promise as a model system for the development of strategies aimed at defining and reducing the CNS reservoir.
Asunto(s)
Complejo SIDA Demencia/tratamiento farmacológico , Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Microglía/efectos de los fármacos , Inhibidores de la Monoaminooxidasa/farmacología , Proteínas del Tejido Nervioso/genética , Fenelzina/farmacología , Proteínas Represoras/genética , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/fisiopatología , Complejo SIDA Demencia/virología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/virología , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Encéfalo/virología , Busulfano/toxicidad , Diferenciación Celular , Proteínas Co-Represoras , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , VIH-1/patogenicidad , VIH-1/fisiología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Ratones , Ratones Transgénicos , Microglía/metabolismo , Microglía/virología , Proteínas del Tejido Nervioso/metabolismo , Ratas , Proteínas Represoras/metabolismo , Trasplante Heterólogo , Latencia del Virus/efectos de los fármacos , Latencia del Virus/genética , Irradiación Corporal TotalRESUMEN
HIV/AIDS has long been at the forefront of the development of gene- and cell-based therapies. Although conventional gene therapy approaches typically involve the addition of anti-HIV genes to cells using semirandomly integrating viral vectors, newer genome editing technologies based on engineered nucleases are now allowing more precise genetic manipulations. The possible outcomes of genome editing include gene disruption, which has been most notably applied to the CCR5 coreceptor gene, or the introduction of small mutations or larger whole gene cassette insertions at a targeted locus. Disruption of CCR5 using zinc finger nucleases was the first-in-human application of genome editing and remains the most clinically advanced platform, with 7 completed or ongoing clinical trials in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we review the laboratory and clinical findings of CCR5 editing in T cells and HSPCs for HIV therapy and summarize other promising genome editing approaches for future clinical development. In particular, recent advances in the delivery of genome editing reagents and the demonstration of highly efficient homology-directed editing in both T cells and HSPCs are expected to spur the development of even more sophisticated applications of this technology for HIV therapy.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Mutagénesis Insercional , Receptores CCR5 , Linfocitos T/metabolismo , Reparación del Gen Blanco/métodos , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Síndrome de Inmunodeficiencia Adquirida/terapia , Desoxirribonucleasas/genética , Humanos , Receptores CCR5/genética , Receptores CCR5/metabolismoRESUMEN
UNLABELLED: Certain members of the Arenaviridae family are category A agents capable of causing severe hemorrhagic fevers in humans. Specific antiviral treatments do not exist, and the only commonly used drug, ribavirin, has limited efficacy and can cause severe side effects. The discovery and development of new antivirals are inhibited by the biohazardous nature of the viruses, making them a relatively poorly understood group of human pathogens. We therefore adapted a reverse-genetics minigenome (MG) rescue system based on Junin virus, the causative agent of Argentine hemorrhagic fever, for high-throughput screening (HTS). The MG rescue system recapitulates all stages of the virus life cycle and enables screening of small-molecule libraries under biosafety containment level 2 (BSL2) conditions. The HTS resulted in the identification of four candidate compounds with potent activity against a broad panel of arenaviruses, three of which were completely novel. The target for all 4 compounds was the stage of viral entry, which positions the compounds as potentially important leads for future development. IMPORTANCE: The arenavirus family includes several members that are highly pathogenic, causing acute viral hemorrhagic fevers with high mortality rates. No specific effective treatments exist, and although a vaccine is available for Junin virus, the causative agent of Argentine hemorrhagic fever, it is licensed for use only in areas where Argentine hemorrhagic fever is endemic. For these reasons, it is important to identify specific compounds that could be developed as antivirals against these deadly viruses.
Asunto(s)
Antivirales/farmacología , Infecciones por Arenaviridae/prevención & control , Arenavirus/fisiología , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Internalización del Virus/efectos de los fármacos , Antivirales/aislamiento & purificación , Humanos , Virus Junin/genética , Genética Inversa/métodosRESUMEN
BACKGROUND: The interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity. RESULTS: By examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin. CONCLUSIONS: These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.
Asunto(s)
Antígenos CD/química , Antígenos CD/metabolismo , VIH-2/genética , VIH-2/metabolismo , Interacciones Huésped-Patógeno/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencias de Aminoácidos , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , VIH-2/inmunología , Humanos , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas Reguladoras y Accesorias Virales/metabolismo , Virión , Replicación Viral , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.
Asunto(s)
Endonucleasas/genética , Genómica/métodos , Células Madre Hematopoyéticas/citología , Receptores CCR5/genética , Dedos de Zinc/genética , Síndrome de Inmunodeficiencia Adquirida/terapia , Adenoviridae/genética , Animales , Antígenos CD34/genética , Antígenos CD34/metabolismo , Apoptosis , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Endonucleasas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Eliminación de Gen , Marcación de Gen , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , VIH-1 , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Receptores CCR5/metabolismoRESUMEN
Genetic strategies to block expression of CCR5, the major co-receptor of human immunodeficiency virus type 1 (HIV-1), are being developed as anti-HIV therapies. For example, human hematopoietic stem/precursor cells (HSPC) can be modified by the transient expression of CCR5-targeted zinc finger nucleases (ZFNs) to generate CCR5-negative cells, which could then give rise to HIV-resistant mature CD4(+) T cells following transplantation into patients. The safety and anti-HIV effects of such treatments can be evaluated by transplanting ZFN-treated HSPC into immunodeficient mice, where the extent of human cell engraftment, lineage differentiation and anti-HIV activity arising from the engineered HSPC can be examined. In this way, humanized mice are providing a powerful small animal model for pre-clinical studies of novel anti-HIV therapies.
Asunto(s)
Terapia Biológica/métodos , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH-1/fisiología , Células Madre Hematopoyéticas/virología , Receptores CCR5/genética , Receptores del VIH/genética , Animales , Terapia Biológica/efectos adversos , Humanos , Ratones , Ratones SCID , Receptores CCR5/deficiencia , Receptores del VIH/deficiencia , Dedos de ZincRESUMEN
The immunoglobulin locus of B cells can be reprogrammed by genome editing to produce custom or non-natural antibodies that are not induced by immunization. However, current strategies for antibody reprogramming require complex expression cassettes and do not allow for customization of the constant region of the antibody. Here we show that human B cells can be edited at the immunoglobulin heavy-chain locus to express heavy-chain-only antibodies that support alterations to both the fragment crystallizable domain and the antigen-binding domain, which can be based on both antibody and non-antibody components. Using the envelope protein (Env) from the human immunodeficiency virus as a model antigen, we show that B cells edited to express heavy-chain antibodies to Env support the regulated expression of B cell receptors and antibodies through alternative splicing and that the cells respond to the Env antigen in a tonsil organoid model of immunization. This strategy allows for the reprogramming of human B cells to retain the potential for in vivo amplification while producing molecules with flexibility of composition beyond that of standard antibodies.
RESUMEN
BST-2/tetherin is an interferon-inducible host restriction factor that blocks the release of newly formed enveloped viruses. It is enriched in lipid raft membrane microdomains, which are also the sites of assembly of several enveloped viruses. Viral anti-tetherin factors, such as the HIV-1 Vpu protein, typically act by removing tetherin from the cell surface. In contrast, the Ebola virus glycoprotein (GP) is unusual in that it blocks tetherin restriction without apparently altering its cell surface localization. We explored the possibility that GP acts to exclude tetherin from the specific sites of virus assembly without overtly removing it from the cell surface and that lipid raft exclusion is the mechanism involved. However, we found that neither GP nor Vpu had any effect on tetherin's distribution within lipid raft domains. Furthermore, GP did not prevent the colocalization of tetherin and budding viral particles. Contrary to previous reports, we also found no evidence that GP is itself a raft protein. Together, our data indicate that the exclusion of tetherin from lipid rafts is not the mechanism used by either HIV-1 Vpu or Ebola virus GP to counteract tetherin restriction.
Asunto(s)
Antígenos CD/metabolismo , Ebolavirus/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Fiebre Hemorrágica Ebola/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Microdominios de Membrana/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Antígenos CD/genética , Línea Celular , Ebolavirus/genética , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Fiebre Hemorrágica Ebola/virología , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Membrana Dobles de Lípidos/metabolismo , Microdominios de Membrana/virología , Unión Proteica , Transporte de Proteínas , Proteínas del Envoltorio Viral/genética , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders caused by mutations in the human type VII collagen gene (COL7A1). Individuals with DEB lack type VII collagen and anchoring fibrils, structures that attach epidermis and dermis. The current lack of treatment for DEB is an impetus to develop gene therapy strategies that efficiently transfer and stably express genes delivered to skin cells in vivo. In this study, we delivered and expressed full-length type VII collagen using a self-inactivating minimal lentivirus-based vector. Transduction of lentiviral vectors containing the COL7A1 transgene into recessive DEB (RDEB) keratinocytes and fibroblasts (in which type VII collagen was absent) resulted in persistent synthesis and secretion of type VII collagen. Unlike RDEB parent cells, the gene-corrected cells had normal morphology, proliferative potential, matrix attachment and motility. We used these gene-corrected cells to regenerate human skin on immune-deficient mice. Human skin regenerated by gene-corrected RDEB cells had restored expression of type VII collagen and formation of anchoring fibrils at the dermal-epidermal junction in vivo. These studies demonstrate that it is possible to restore type VII collagen gene expression in RDEB skin in vivo.
Asunto(s)
Colágeno Tipo VII/genética , Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/metabolismo , Adhesión Celular , División Celular , Línea Celular , Movimiento Celular , Transformación Celular Viral , Células Cultivadas , Colágeno Tipo VII/biosíntesis , ADN Complementario , Células Epidérmicas , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/terapia , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/ultraestructura , Técnicas de Transferencia de Gen , Genes Recesivos , Terapia Genética , Vectores Genéticos , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/ultraestructura , Laminina/metabolismo , Lentivirus/genética , Mutación , Transfección , TransgenesRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a SARS-like coronavirus, continues to produce mounting infections and fatalities all over the world. Recent data point to SARS-CoV-2 viral infections in the human testis. As low testosterone levels are associated with SARS-CoV-2 viral infections in males and human Leydig cells are the main source of testosterone, we hypothesized that SARS-CoV-2 could infect human Leydig cells and impair their function. We successfully detected SARS-CoV-2 nucleocapsid in testicular Leydig cells of SARS-CoV-2-infected hamsters, providing evidence that Leydig cells can be infected with SARS-CoV-2. We then employed human Leydig-like cells (hLLCs) to show that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 is highly expressed in hLLCs. Using a cell binding assay and a SARS-CoV-2 spike-pseudotyped viral vector (SARS-CoV-2 spike pseudovector), we showed that SARS-CoV-2 could enter hLLCs and increase testosterone production by hLLCs. We further combined the SARS-CoV-2 spike pseudovector system with pseudovector-based inhibition assays to show that SARS-CoV-2 enters hLLCs through pathways distinct from those of monkey kidney Vero E6 cells, a typical model used to study SARS-CoV-2 entry mechanisms. We finally revealed that neuropilin-1 and cathepsin B/L are expressed in hLLCs and human testes, raising the possibility that SARS-CoV-2 may enter hLLCs through these receptors or proteases. In conclusion, our study shows that SARS-CoV-2 can enter hLLCs through a distinct pathway and alter testosterone production.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Masculino , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Testosterona/metabolismo , Células Intersticiales del Testículo/metabolismo , Testículo/metabolismo , Peptidil-Dipeptidasa A/metabolismoRESUMEN
We describe a genome editing strategy to reprogram the immunoglobulin heavy chain (IgH) locus of human B cells to express custom molecules that respond to immunization. These heavy chain antibodies (HCAbs) comprise a custom antigen-recognition domain linked to an Fc domain derived from the IgH locus and can be differentially spliced to express either B cell receptor (BCR) or secreted antibody isoforms. The HCAb editing platform is highly flexible, supporting antigen-binding domains based on both antibody and non-antibody components, and also allowing alterations in the Fc domain. Using HIV Env protein as a model antigen, we show that B cells edited to express anti-Env HCAbs support the regulated expression of both BCRs and antibodies, and respond to Env antigen in a tonsil organoid model of immunization. In this way, human B cells can be reprogrammed to produce customized therapeutic molecules with the potential for in vivo amplification.