FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

SARS-CoV-2 Evolution: On the Sudden Appearance of the Omicron Variant.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and harbors many mutations in the spike protein, but the origin of this virus variant remains unclear. We address the role of unusual virus evolution mechanisms such as hypermutation, out-of-frame reading, and recombination. Rather, regular Darwinian evolution, that is, the repeated selection of beneficial spike mutations, seems to have led to the appearance of the grossly altered spike protein of the Omicron variant.

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates.

BACKGROUND: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. METHODS: Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. RESULTS: Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. CONCLUSIONS: These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.

A CRISPR-Cas Cure for HIV/AIDS.

Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure.

Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity.

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).

Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA.

The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.

Boosting AgoshRNA activity by optimized 5'-terminal nucleotide selection.

RNA interference (RNAi) can be triggered by synthetic small interfering RNAs (siRNAs) or transgene-expressed short hairpin RNAs (shRNAs). Recent evidence indicates that shRNA molecules, with a relatively short stem and small loop, are processed by Argonaute 2 protein (Ago2). We named these molecules AgoshRNA as Ago2 is involved in both the processing and the subsequent mRNA-silencing reaction. This alternative processing route yields only a single guide strand, which thus avoids potential off-target effects induced by the passenger strand of a regular shRNA. We recently described that the introduction of a 5'-terminal purine (A or G) and a mismatch at the bottom of the hairpin enhances the AgoshRNA activity. The critical 5'-terminal nucleotide (nt) represents the +1 position of the transcriptional promoter, which influences the transcriptional efficiency and initiation accuracy as demonstrated for the H1 RNA polymerase (Pol) III promoter. These findings highlight the necessity of considering Pol III requirements in the design of optimized AgoshRNA cassettes. In this study, we report the design and expression of potent AgoshRNAs by two other popular Pol III promoters: U6 and 7SK, which were recently reported to have a distinct transcription profile compared to the H1 promoter. We propose general rules for the design and expression of potent AgoshRNA molecules using Pol III cassettes, which should augment the application of novel AgoshRNA reagents for basic research and therapeutic purposes.

Dicer-independent processing of small RNA duplexes: mechanistic insights and applications.

MicroRNAs (miRNAs) play a pivotal role in the regulation of cellular gene expression via the conserved RNA interference (RNAi) mechanism. Biogenesis of the unusual miR-451 does not require Dicer. This molecule is instead processed by the Argonaute 2 (Ago2) enzyme. Similarly, unconventional short hairpin RNA (shRNA) molecules have been designed as miR-451 mimics that rely exclusively on Ago2 for maturation. We will review recent progress made in the understanding of this alternative processing route. Next, we describe different Dicer-independent shRNA designs that have been developed and discuss their therapeutic advantages and disadvantages. As an example, we will present the route towards development of a durable gene therapy against HIV-1.

The influence of the 5΄-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site.

Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecules.

Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA.

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of self-cleaving ribozymes that facilitate processing of the crRNA transcript to produce the precise guide molecule. Insertion of the 3'-terminal HDV ribozyme boosted the gene editing activity of the CRISPR-Cpf1 system ranging from 1.1 to 5.2 fold. We also demonstrate that this design can enhance CRISPR-based gene activation. We thus generated an improved CRISPR-Cpf1 system for more efficient gene editing and gene regulation.

Influence of the loop size and nucleotide composition on AgoshRNA biogenesis and activity.

Short hairpin RNAs (shRNAs) are widely used for gene silencing by the RNA interference (RNAi) mechanism. The shRNA precursor is processed by the Dicer enzyme into active small interfering RNAs (siRNAs) that subsequently target a complementary mRNA for cleavage by the Argonaute 2 (Ago2) complex. Recent evidence indicates that shRNAs with a relatively short basepaired stem bypass Dicer and are instead processed by Ago2. We termed these molecules AgoshRNAs as both processing and silencing steps are mediated by Ago2 and proposed rules for the design of effective AgoshRNA molecules. Active and non-cytotoxic AgoshRNAs against HIV-1 RNA were generated, but their silencing activity was generally reduced compared with the matching shRNAs. Thus, further optimization of the AgoshRNA design is needed. In this study, we evaluated the importance of the single-stranded loop, in particular its size and nucleotide sequence, in AgoshRNA-mediated silencing. We document that the pyrimidine/purine content is important for AgoshRNA-mediated silencing activity.

Probing the shRNA characteristics that hinder Dicer recognition and consequently allow Ago-mediated processing and AgoshRNA activity.

Recent evidence indicates the presence of alternative pathways for microRNA (miRNA) and short hairpin (shRNA) processing. Specifically, some of these molecules are refractory to Dicer-mediated processing, which allows alternative processing routes via the Ago2 endonuclease. The resulting RNA molecules differ in size and sequence and will thus trigger the silencing of different target RNAs. It is, therefore, important to understand these processing routes in mechanistic detail such that one can design exclusive RNA reagents for a specific processing route. The exact sh/miRNA properties that determine this routing toward Dicer or Ago2 are incompletely understood. The size of the base-paired stem seems an important determinant, but other RNA elements may contribute as well. In this study, we document the importance of a weak G-U or U-G base pair at the top of the hairpin stem.

Attacking HIV-1 RNA versus DNA by sequence-specific approaches: RNAi versus CRISPR-Cas.

Human immunodeficiency virus type 1 (HIV-1) infection can be effectively controlled by potent antiviral drugs, but this never results in a cure. The patient should therefore take these drugs for the rest of his/her life, which can cause drug-resistance and adverse effects. Therefore, more durable therapeutic strategies should be considered, such as a stable gene therapy to protect the target T cells against HIV-1 infection. The development of potent therapeutic regimens based on the RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) mechanisms will be described, which can be delivered by lentiviral vectors. These mechanisms attack different forms of the viral genome, the RNA and DNA, respectively, but both mechanisms act in a strictly sequence-specific manner. Early RNAi experiments demonstrated profound virus inhibition, but also indicated that viral escape is possible. Such therapy failure can be prevented by the design of a combinatorial RNAi attack on the virus and this gene therapy is currently being tested in a preclinical humanized mouse model. Recent CRISPR-Cas studies also document robust virus inhibition, but suggest a novel viral escape route that is induced by the cellular nonhomologous end joining DNA repair pathway, which is activated by CRISPR-Cas-induced DNA breaks. We will compare these two approaches for durable HIV-1 suppression and discuss the respective advantages and disadvantages. The potential for future clinical applications will be described.

Toward optimization of AgoshRNA molecules that use a non-canonical RNAi pathway: variations in the top and bottom base pairs.

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism. The shRNA precursor is processed by Dicer into small interfering RNAs (siRNAs) and subsequently programs the RNAi-induced silencing complex (RISC) to find a complementary target mRNA (mRNA) for post-transcriptional gene silencing. Recent evidence indicates that shRNAs with a relatively short basepaired stem bypass Dicer to be processed directly by the Ago2 nuclease of the RISC complex. We named this design AgoshRNA as these molecules depend on Ago2 both for processing and subsequent silencing activity. This alternative AgoshRNA processing route yields only a single active RNA strand, an important feature to restrict off-target effects induced by the passenger strand of regular shRNAs. It is therefore important to understand this novel AgoshRNA processing route in mechanistic detail such that one can design the most effective and selective RNA reagents. We performed a systematic analysis of the optimal base pair (bp) composition at the top and bottom of AgoshRNA molecules. In this study, we document the importance of the 5' end nucleotide (nt) and a bottom mismatch. The optimized AgoshRNA design exhibits improved RNAi activity across cell types. These results have important implications for the future design of more specific RNAi therapeutics.

Mechanistic insights on the Dicer-independent AGO2-mediated processing of AgoshRNAs.

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both for research and therapeutic purposes. The shRNA precursor is processed by the RNase III-like enzyme Dicer into biologically active small interfering RNA (siRNA). This effector molecule subsequently targets a complementary mRNA for destruction via the Argonaute 2 (AGO2) complex. The cellular role of Dicer concerns the processing of pre-miRNAs into mature microRNA (miRNA). Recently, a non-canonical pathway was reported for the biogenesis of miR-451, which bypasses Dicer and is processed instead by the slicer activity of AGO2, followed by the regular AGO2-mediated mRNA targeting step. Interestingly, shRNA designs that are characterized by a relatively short basepaired stem also bypass Dicer to be processed by AGO2. We named this design AgoshRNA as these molecules depend on AGO2 both for processing and silencing activity. In this study, we investigated diverse mechanistic aspects of this new class of AgoshRNA molecules. We probed the requirements for AGO2-mediated processing of AgoshRNAs by modification of the proposed cleavage site in the hairpin. We demonstrate by deep sequencing that AGO2-processed AgoshRNAs produce RNA effector molecules with more discrete ends than the products of the regular shRNA design. Furthermore, we tested whether trimming and tailing occurs upon AGO2-mediated processing of AgoshRNAs, similar to what has been described for miR-451. Finally, we tested the prediction that AgoshRNA activity, unlike that of regular shRNAs, is maintained in Dicer-deficient cell types. These mechanistic insights could aid in the design of optimised AgoshRNA tools and therapeutics.

The impact of unprotected T cells in RNAi-based gene therapy for HIV-AIDS.

RNA interference (RNAi) is highly effective in inhibiting human immunodeficiency virus type 1 (HIV-1) replication by the expression of antiviral short hairpin RNA (shRNA) in stably transduced T-cell lines. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against highly conserved regions of the HIV-1 RNA genome. The future in vivo application of such a gene therapy protocol will reach only a fraction of the T cells, such that HIV-1 replication will continue in the unmodified T cells, thereby possibly frustrating the therapy by generation of HIV-1 variants that escape from the inhibition imposed by the protected cells. We studied virus inhibition and evolution in pure cultures of shRNA-expressing cells versus mixed cell cultures of protected and unprotected T cells. The addition of the unprotected T cells indeed seems to accelerate HIV-1 evolution and escape from a single shRNA inhibitor. However, expression of three antiviral shRNAs from a single lentiviral vector prevents virus escape even in the presence of unprotected cells. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models.

Gene therapy strategies to block HIV-1 replication by RNA interference.

The cellular mechanism of RNA interference (RNAi) plays an antiviral role in many organisms and can be used for the development of therapeutic strategies against viral pathogens. Persistent infections like the one caused by the human immunodeficiency virus type 1 (HIV-1) likely require a durable gene therapy approach. The continuous expression of the inhibitory RNA molecules in T cells is needed to effectively block HIV-1 replication. We discuss here several issues, ranging from the choice of RNAi inhibitor and vector system, finding the best target in the HIV-1 RNA genome, alternatively by targeting host mRNAs that encode important viral cofactors, to the setup of appropriate preclinical test systems. Finally, we briefly discuss the relevance of this topic for other viral pathogens that cause a chronic infection in humans.

Evaluation of the effect of RNA secondary structure on Cas13d-mediated target RNA cleavage.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. A comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structures than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.

CRISPR-Cas12b enables a highly efficient attack on HIV proviral DNA in T cell cultures.

BACKGROUND: The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a. METHODS: We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells. FINDINGS: We demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such "hypermutated" HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation. INTERPRETATION: These results provide in vitro "proof of concept' for CRISPR-Cas12b mediated HIV-1 inactivation.

Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2.

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus -RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals.