Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides.

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.

Cytoplasmic Tail Truncation of SARS-CoV-2 Spike Protein Enhances Titer of Pseudotyped Vectors but Masks the Effect of the D614G Mutation.

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.

Genome edited B cells: a new frontier in immune cell therapies.

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.

Homologous Recombination-Based Genome Editing by Clade F AAVs Is Inefficient in the Absence of a Targeted DNA Break.

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.

Plasmacytoid and conventional dendritic cells cooperate in crosspriming AAV capsid-specific CD8+ T cells.

Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8+ T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8+ T cells against the input viral capsid antigen. In this study, we report that the Toll-like receptor 9 (TLR9)-MyD88 pattern-recognition receptor pathway is uniquely capable of initiating this response. By contrast, the absence of TLR2, STING, or the addition of TLR4 agonist has no effect. Surprisingly, both conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are required for the crosspriming of capsid-specific CD8+ T cells, whereas other antigen-presenting cells are not involved. TLR9 signaling is specifically essential in pDCs but not in cDCs, indicating that sensing of the viral genome by pDCs activates cDCs in trans to cross-present capsid antigen during CD8+ T-cell activation. Cross-presentation and crosspriming depend not only on TLR9, but also on interferon type I signaling, and both mechanisms can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8+ T cells. Thus, these outcomes directly point to therapeutic interventions and demonstrate that innate immune blockade can eliminate unwanted immune responses in gene therapy.

Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells.

Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80(+) cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103(+) and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4(+) regulatory T cells (CD4(+)CD25(-)LAP(+) cells with upregulated IL-10 and transforming growth factor-ß (TGF-ß) expression) as well as conventional CD4(+)CD25(+) regulatory T cells systemically suppressed anti-FIX responses.

Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell-dependent induction of CD4+CD25+FoxP3+ Treg.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg) are critical elements for maintaining immune tolerance, for instance to exogenous antigens that are introduced during therapeutic interventions such as cell/organ transplant or gene/protein replacement therapy. Coadministration of antigen with rapamycin simultaneously promotes deletion of conventional CD4(+) T cells and induction of Treg. Here, we report that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin. This occurs via selective expansion of plasmacytoid dendritic cells (pDCs), which further augments the number of Treg. Whereas in conventional DCs, rapamycin effectively blocks mammalian target of rapamycin (mTOR) 1 signaling induced by Flt3L, increased mTOR1 activity renders pDCs more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDCs. This concept is supported by the finding that Treg induction is abrogated upon pDC depletion. The combination with pDCs and rapamycin is requisite for Flt3L/antigen-induced Treg induction because Flt3L/antigen by itself fails to induce Treg. As co-administering Flt3L, rapamycin, and antigen blocked CD8(+) T-cell and antibody responses in models of gene and protein therapy, we conclude that the differential effect of rapamycin on DC subsets can be exploited for improved tolerance induction.

Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B.

BACKGROUND: Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX). METHODS: Hemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8⁺ T cell responses to hF.IX. RESULTS: Self-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8⁺ T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome. CONCLUSIONS: The nature of the AAV genome can impact the CD8⁺ T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome.

Differential type I interferon-dependent transgene silencing of helper-dependent adenoviral vs. adeno-associated viral vectors in vivo.

We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection. This type I IFN signaling in response to HDAds administration contributes to transcriptional silencing of both HDAd prokaryotic and eukaryotic DNA in liver. This silencing occurs early and is mediated by epigenetic modification as shown by in vivo chromatin immunoprecipitation (ChIP) with anti-histone deacetylase (HDAC) and promyelocytic leukemia protein (PML). In contrast, self-complementary adeno-associated viral vectors (scAAVs) showed significantly lower induction of type I IFN mRNA in liver compared to HDAds at both early and late time points. These results show that the type I IFN signaling dependent transgene silencing differs between AAV and HDAd vectors after liver-directed gene transfer.

Reprogramming human B cells with custom heavy-chain antibodies.

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.

Reprogramming human B cells with custom heavy chain antibodies.

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.

Reprogramming human B cells with custom heavy chain antibodies.

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.

Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes.

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold, respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50- to 100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.

Gene Therapy Approaches to Human Immunodeficiency Virus and Other Infectious Diseases.

Advances in gene therapy technologies, particularly in gene editing, are suggesting new avenues for the treatment of human immunodeficiency virus and other infectious diseases. This article outlines recent developments in antiviral gene therapies, including those based on the disruption of entry receptors or that target viral genomes using targeted nucleases, such as the CRISPR/Cas9 system. In addition, new ways to express circulating antiviral factors, such as antibodies, and approaches to harness and engineer the immune system to provide an antiviral effect that is not naturally achieved are described.

Combination therapy for inhibitor reversal in haemophilia A using monoclonal anti-CD20 and rapamycin.

Development of antibodies (inhibitors) against coagulation factor VIII (FVIII) is a major complication of intravenous replacement therapy in haemophilia A (HA). Current immune tolerance induction (ITI) regimens are not universally effective. Rituximab, a B cell-depleting antibody against CD20, has shown mixed results for inhibitor reversal in patients. This study aims to develop a combinatorial therapy for inhibitor reversal in HA, using anti-murine CD20 (anti-mCD20) antibody and rapamycin, which targets both B and T cell responses. Additionally, it extensively characterises the role of the IgG backbone in B cell depletion by anti-CD20 antibodies. For this, inhibitors were generated in BALB/c-HA mice by weekly IV injection of FVIII. Subsequently, anti-mCD20 (18B12) with IgG2a or IgG1 backbone was injected IV in two doses three weeks apart and B cell depletion and recovery was characterised. Rapamycin was administered orally 3x/week (for 1 month) while continuing FVIII injections. Altering the IgG backbone of anti-mCD20 from IgG2a to IgG1 reduced overall depletion of B cells (including memory B cells), and marginal zone, B-10, and B-1b cells were specifically unaffected. While neither antibody was effective alone, in combination with rapamycin, anti-mCD20 IgG2a but not IgG1 was able to reverse inhibitors in HA mice. This regimen was particularly effective for starting titres of ~10 BU. Although IgG1 anti-mCD20 spared potentially tolerogenic B cell subsets, IgG2a directed sustained hyporesponsiveness when administered in conjunction with rapamycin. This regimen represents a promising treatment for inhibitor reversal in HA, as both of these compounds have been extensively used in human patients.

Gene therapy for hemophilia.

Hemophilia is an X-linked inherited bleeding disorder consisting of two classifications, hemophilia A and hemophilia B, depending on the underlying mutation. Although the disease is currently treatable with intravenous delivery of replacement recombinant clotting factor, this approach represents a significant cost both monetarily and in terms of quality of life. Gene therapy is an attractive alternative approach to the treatment of hemophilia that would ideally provide life-long correction of clotting activity with a single injection. In this review, we will discuss the multitude of approaches that have been explored for the treatment of both hemophilia A and B, including both in vivo and ex vivo approaches with viral and nonviral delivery vectors.

Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer.

The immune system represents a significant barrier to successful gene therapy with adeno-associated viral (AAV) vectors. In particular, adaptive immune responses to the viral capsid or the transgene product are of concern. The sensing of AAV by toll-like receptors (TLRs) TLR2 and TLR9 has been suggested to play a role in innate immunity to the virus and may also shape subsequent adaptive immune responses. Here, we investigated the functions of TLR2, TLR9 and the downstream signaling adaptor MyD88 in antibody and CD8+ T-cell responses. Antibody formation against the transgene product occurred largely independently of TLR signaling following gene transfer with AAV1 or AAV2 vectors, whereas loss of signaling through the TLR9-MyD88 pathway substantially reduced CD8+ T-cell responses. In contrast, MyD88 (but neither of the TLRs) regulated antibody responses to capsid. B cell-intrinsic MyD88 was required for the formation of anti-capsid IgG2c independently of vector serotype or route of administration. However, MyD88(-/-) mice instead produced anti-capsid IgG1 that emerged with delayed kinetics but nonetheless completely prevented in vivo readministration. We conclude that there are distinct roles for TLR9 and MyD88 in promoting adaptive immune responses to AAV-mediated gene transfer and that there are redundant MyD88-dependent and MyD88-independent mechanisms that stimulate neutralizing antibody formation against AAV.