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Extracellular vesicles (EVs) are considered a vital component of cell-to-cell communication and represent a new frontier in diagnostics and a means to identify pathways for therapeutic intervention. Recently, studies have revealed the importance of tissue-derived EVs (Ti-EVs), which are EVs present in the interstitial spaces between cells, as they better represent the underlying physiology of complex, multicellular tissue microenvironments in biology and disease. EVs are native, lipid bilayer membraned nano-sized particles produced by all cells that are packaged with varied functional biomolecules including proteins, lipids, and nucleic acids. They are implicated in short- and long-range cellular communication and may elicit functional responses in recipient cells. To date, studies have often utilized cultured cells or biological fluids as a source for EVs that do not capture local molecular signatures of the tissue microenvironment. Recent work utilizing Ti-EVs has elucidated novel biomarkers for disease and provided insights into disease mechanisms that may lead to the development of novel therapeutic agents. Still, there are considerable challenges facing current studies. This review explores the vast potential and unique challenges for Ti-EV research and provides considerations for future studies that seek to advance this exciting field.
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Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.
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Tratamiento Farmacológico de COVID-19 , Sistemas de Liberación de Medicamentos/métodos , Lípidos/química , Nanopartículas/química , ARN Bicatenario/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , SARS-CoV-2/genética , Administración Intravenosa , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/metabolismo , COVID-19/virología , Femenino , Silenciador del Gen , Células HEK293 , Humanos , Pulmón/metabolismo , Masculino , Ratones , Ratones Transgénicos , ARN Bicatenario/genética , ARN Viral/genética , Transcriptoma/efectos de los fármacos , Resultado del TratamientoRESUMEN
Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.
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Carcinogénesis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/virología , Virus Oncogénicos/genética , Animales , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Viral/efectos de los fármacos , Transformación Celular Viral/genética , Humanos , Ratones , Oncogenes , Virus Oncogénicos/patogenicidadRESUMEN
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Terapia Molecular Dirigida/métodos , Mucosa Nasal/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular , Membrana Celular/metabolismo , Péptidos de Penetración Celular/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/terapia , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Nanopartículas/química , Mucosa Nasal/citología , ARN Guía de Kinetoplastida/farmacología , ARN Largo no Codificante/genética , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo. RESULTS: Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state. CONCLUSIONS: Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of "RNA Machines" that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.
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Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/genética , ARN no Traducido/genética , Transcripción Genética , Antirretrovirales/farmacología , Biomimética , Sistemas CRISPR-Cas , Línea Celular , Edición Génica , Silenciador del Gen , VIH-1/efectos de los fármacos , Humanos , Regiones Promotoras Genéticas , ARN Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The "shock and kill" strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a "hotspot" for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a "functional cure" of HIV/AIDS.
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Sistemas CRISPR-Cas , VIH-1/fisiología , Complejos Multiproteicos/metabolismo , Activación Viral , Latencia del Virus , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/metabolismo , Regulación Viral de la Expresión Génica , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH/genética , Humanos , FN-kappa B/metabolismo , Motivos de Nucleótidos , Unión Proteica , ARN Guía de Kinetoplastida/genética , Activación TranscripcionalRESUMEN
Human T-lymphotropic virus type I (HTLV-I) infects CD4+ T-cells resulting in a latent, life-long infection in patients. Crosstalk between oncogenic viral factors results in the transformation of the host cell into an aggressive cancer, adult T-cell leukemia/lymphoma (ATL). ATL has a poor prognosis with no currently available effective treatments, urging the development of novel therapeutic strategies. Recent evidence exploring those mechanisms contributing to ATL highlights the viral anti-sense gene HTLV-I bZIP factor (HBZ) as a tumor driver and a potential therapeutic target. In this work, a series of zinc-finger protein (ZFP) repressors were designed to target within the HTLV-I promoter that drives HBZ expression at highly conserved sites covering a wide range of HTLV-I genotypes. ZFPs were identified that potently suppressed HBZ expression and resulted in a significant reduction in the proliferation and viability of a patient-derived ATL cell line with the induction of cell cycle arrest and apoptosis. These data encourage the development of this novel ZFP strategy as a targeted modality to inhibit the molecular driver of ATL, a possible next-generation therapeutic for aggressive HTLV-I associated malignancies.
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Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy.
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One of the greatest challenges in the treatment of cancer is tumor heterogeneity which results in differential responses to chemotherapy and drugs that work through a single pathway. A therapeutic agent that targets cancer cells for death through multiple mechanisms could be advantageous as a broad inhibitor for many types of cancers and the heterogeneous alterations they possess. Several viral proteins have been exploited for antiproliferative and apoptotic effect in cancer cells by disrupting critical survival pathways. Here, we report the use of the non-structural protein on the S segment (NSs) gene from the Rift Valley fever virus (RVFV) to induce cancer cell death. NSs has immune evasion functions in the context of RVFV with many of these functions affecting proliferation pathways and DNA damage signaling, which could be leveraged against cancer cells. We find that expression of NSs in multiple cancer cell lines leads to a rapid decline in cell viability and induction of apoptosis. Interestingly, we observed reduced toxicity in normal cells suggesting cancer cells may be more susceptible to NSs-mediated cell death. To enhance specificity of NSs for use in hepatocellular carcinoma, we incorporated four miR-122 binding sites in the 3' untranslated region (UTR) of the NSs mRNA to achieve cell type specific expression. Observations presented here collectively suggest that delivery of the NSs gene may provide a unique therapeutic approach in a broad range of cancers.
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MicroARNs , Neoplasias , Virus de la Fiebre del Valle del Rift , Regiones no Traducidas 3' , Animales , Genes Relacionados con las Neoplasias , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias/genética , Neoplasias/terapia , Virus de la Fiebre del Valle del Rift/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19, urging the development of novel modalities. Here, we describe a molecular therapy specifically targeted to neutralize SARS-CoV-2, which consists of extracellular vesicles (EVs) containing a novel fusion tetraspanin protein, CD63, embedded within an anti-CoV-2 nanobody. These anti-CoV-2-enriched EVs bind SARS-CoV-2 spike protein at the receptor-binding domain (RBD) site and can functionally neutralize SARS-CoV-2. This work demonstrates an innovative EV-targeting platform that can be employed to target and inhibit the early stages of SARS-CoV-2 infection.
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T cells engineered to express HIV-specific chimeric antigen receptors (CARs) represent a promising strategy to clear HIV-infected cells, but to date have not achieved clinical benefits. A likely hurdle is the limited T cell activation and persistence when HIV antigenemia is low, particularly during antiretroviral therapy (ART). To overcome this issue, we propose to use a cytomegalovirus (CMV) vaccine to stimulate CMV-specific T cells that express CARs directed against the HIV-1 envelope protein gp120. In this study, we use a GMP-compliant platform to engineer CMV-specific T cells to express a second-generation CAR derived from the N6 broadly neutralizing antibody, one of the broadest anti-gp120 neutralizing antibodies. These CMV-HIV CAR T cells exhibit dual effector functions upon in vitro stimulation through their endogenous CMV-specific T cell receptors or the introduced CARs. Using a humanized HIV mouse model, we show that CMV vaccination during ART accelerates CMV-HIV CAR T cell expansion in the peripheral blood and that higher numbers of CMV-HIV CAR T cells were associated with a better control of HIV viral load and fewer HIV antigen p24+ cells in the bone marrow upon ART interruption. Collectively, these data support the clinical development of CMV-HIV CAR T cells in combination with a CMV vaccine in HIV-infected individuals.
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Cystic fibrosis is a genetic disorder that results in a multi-organ disease with progressive respiratory decline which leads to premature death. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupts the capacity of the protein to function as a channel, transporting chloride ions and bicarbonate across epithelial cell membranes. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but are only effective for a small percentage of individuals with specific CFTR mutations. To overcome this limitation, we engineered stromal-derived mesenchymal stem cells (MSC) and HEK293 cells to produce exosomes containing a novel CFTR Zinc Finger Protein fusion with transcriptional activation domains VP64, P65 and Rta to target the CFTR promoter (CFZF-VPR) and activate transcription. Treatment with CFZF-VPR results in robust activation of CFTR transcription in patient derived Human Bronchial Epithelial cells (HuBEC). We also find that CFZF-VPR can be packaged into MSC and HEK293 cell exosomes and delivered to HuBEC cells to potently activate CFTR expression. Connexin 43 appeared to be required for functional release of CFZF-VPR from exosomes. The observations presented here demonstrate that MSC derived exosomes can be used to deliver a packaged zinc finger activator to target cells and activate CFTR. The novel approach presented here offers a next-generation genetic therapy that may one day prove effective in treating patients afflicted with Cystic fibrosis.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/terapia , Exosomas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Bicarbonatos/metabolismo , Membrana Celular/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Terapia Genética/métodos , Células HEK293 , Humanos , Transporte Iónico , Terapia Molecular Dirigida/métodos , Dedos de ZincRESUMEN
Human Immunodeficiency Virus (HIV-1) produces a persistent latent infection. Control of HIV-1 using combination antiretroviral therapy (cART) comes at the cost of life-shortening side effects and development of drug-resistant HIV-1. An ideal and safer therapy should be deliverable in vivo and target the stable epigenetic repression of the virus, inducing a stable "block and lock" of virus expression. Towards this goal, we developed an HIV-1 promoter-targeting Zinc Finger Protein (ZFP-362) fused to active domains of DNA methyltransferase 3 A to induce long-term stable epigenetic repression of HIV-1. Cells were engineered to produce exosomes packaged with RNAs encoding this HIV-1 repressor protein. We find here that the repressor loaded anti-HIV-1 exosomes suppress virus expression and that this suppression is mechanistically driven by DNA methylation of HIV-1 in humanized NSG mouse models. The observations presented here pave the way for an exosome-mediated systemic delivery platform of therapeutic cargo to epigenetically repress HIV-1 infection.
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Represión Epigenética/genética , Exosomas/metabolismo , VIH-1/genética , Animales , Encéfalo/patología , Encéfalo/virología , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , ADN Metiltransferasa 3A , Exosomas/ultraestructura , Regulación Viral de la Expresión Génica , Vectores Genéticos/metabolismo , Células HEK293 , Infecciones por VIH/virología , Humanos , Lentivirus/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetidas Terminales/genética , Carga Viral , Dedos de ZincRESUMEN
Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance of HIV-1 infection results in integration of proviral DNA into the genome of host cells, which provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV-1 provirus could be transformative and is the goal for the "shock-and-kill" approach to a functional cure for HIV-1. Substantial progress has been made toward the development of recombinant proteins that target specific genomic loci for gene activation, repression, or inactivation by directed mutations. However, most of these modalities are too large or too complex for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, which specifically and potently enhances proviral HIV-1 transcription both in established latency models and activity across different viral clades. Additionally, ZFP-362-VPR-activated HIV-1 reporter gene expression in a well-established primary human CD4+ T cell latency model and off-target pathways were determined by transcriptome analyses. This study provides clear proof of concept for the application of a novel, therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1.
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Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system. Multiple small-interfering RNAs (siRNAs) targeting highly conserved regions of the SARS-CoV-2 virus were screened and three candidate siRNAs emerged that effectively inhibit virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel lipid nanoparticle formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.
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The RNA-guided, modified type II prokaryotic CRISPR with CRISPR-associated proteins (CRISPR/Cas9) system represents a simple gene-editing platform with applications in biotechnology and also potentially as a therapeutic modality. The system requires a small guide RNA (sgRNA) and a catalytic Cas9 protein to induce non-homologous end joining (NHEJ) at break sites, resulting in the formation of inactivating mutations, or through homology-directed repair (HDR) can engineer in specific sequence changes. Although CRISPR/Cas9 is a powerful technology, the effects can be limited as a result of nuclease-mediated degradation of the RNA components. Significant research has focused on the solid-phase synthesis of CRISPR RNA components with chemically modified bases, but this approach is technically challenging and expensive. Development of a simple, generic approach to generate chemically modified CRISPR RNAs may broaden applications that require nuclease-resistant CRISPR components. We report here the development of a novel, functional U-replaced trans-activating RNA (tracrRNA) that can be in vitro transcribed with chemically stabilizing 2'-fluoro (2'F)-pyrimidines. These data represent a unique and facile approach to generating chemically stabilized CRISPR RNA.
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Human immunodeficiency virus (HIV) is an attractive target for chimeric antigen receptor (CAR) therapy. CAR T cells have proved remarkably potent in targeted killing of cancer cells, and we surmised that CAR T cells could prove useful in eradicating HIV-infected cells. Toward this goal, we interrogate several neutralizing single-chain variable fragments (scFvs) that target different regions of the HIV envelope glycoprotein, gp120. We find here that CAR T cells with scFv from NIH45-46 antibody demonstrated the highest cytotoxicity. Although NIH45-46 CAR T cells are capable of eliminating antigen-expressing cells, we wanted to address HIV reactivation from ex vivo culture of HIV patient-derived CAR T cells. In order to capitalize on the HIV reactivation, we developed a conditionally replicating lentiviral vector (crLV). The crLV can hijack HIV machinery, forming a chimeric lentivirus (LV) instead of HIV and delivered to uninfected cells. We find that CAR T cells generated with crLVs have similar CAR-mediated functionality as traditional CARs. We also demonstrate crLVs' capability of expanding CAR percentage and protecting CD4 CAR T cell in HIV donors. Collectively, we demonstrate here that the novel crLV NIH45-46 CAR can serve as a strategy to combat HIV, as well as overcome HIV reactivation in CD4+ CAR T cells.
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CRISPR/Cas is a transformative gene editing tool, that offers a simple and effective way to target a catalytic Cas9, the most widely used is derived from Streptococcus pyogenes (SpCas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology, and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore, the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines, which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally, we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity.
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Proteína 9 Asociada a CRISPR/metabolismo , Edición Génica/métodos , ARN Guía de Kinetoplastida/genética , Ribonucleoproteínas/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Proteína 9 Asociada a CRISPR/química , Edición Génica/instrumentación , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Humanos , Mutación INDEL , ARN Guía de Kinetoplastida/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Ribonucleoproteínas/genética , Streptococcus pyogenes/enzimologíaRESUMEN
HIV-1 infection continues to be a global health challenge and a vaccine is urgently needed. Broadly neutralizing antibodies (bNAbs) are considered essential as they inhibit multiple HIV-1 strains, but they are difficult to elicit by conventional immunization. In contrast, non-neutralizing antibodies that correlated with reduced risk of infection in the RV144 HIV vaccine trial are relatively easy to induce, but responses are not durable. To overcome these obstacles, adeno-associated virus (AAV) vectors were used to provide long-term expression of antibodies targeting the V2 region of the HIV-1 envelope protein, including the potent CAP256-VRC26.25 bNAb, as well as non-neutralizing CAP228 antibodies that resemble those elicited by vaccination. AAVs mediated effective antibody expression in cell culture and immunocompetent mice. Mean concentrations of human immunoglobulin G (IgG) in mouse sera increased rapidly following a single AAV injection, reaching 8-60 µg/mL for CAP256 antibodies and 44-220 µg/mL for CAP228 antibodies over 24 weeks, but antibody concentrations varied for individual mice. Secreted antibodies collected from serum retained the expected binding and neutralizing activity. The vectors generated here are, therefore, suitable for the delivery of V2-targeting HIV antibodies, and they could be used in a vectored immunoprophylaxis (VIP) approach to sustain the level of antibody expression required to prevent HIV infection.