Gene Delivery Using Baculovirus in Human Hematopoietic Stem and Progenitor Cells Requires Inhibition of Cellular Innate Immune Pathways

Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) has the potential to treat inherited blood disorders not amendable to CRISPR-Cas9 gene inactivation or single base editing. For many diseases, one of the major hurdles is viral delivery of large DNA templates needed for gene correction. Due to limited adeno-associated virus (AAV) packaging capacity other delivery approaches are needed. Baculovirus (BV), specifically Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is a large double-stranded DNA (dsDNA) virus widely used for protein expression and AAV production. In addition, BV has been proposed as a potential therapeutic vector (Ono, Viruses 2018). BV does not replicate in mammalian cells, can deliver large quantities of DNA with virtually unlimited packaging capacity, and can express genes under the control of mammalian promoters. While capable of transducing human hepatic cells and some cell lines (Chen, Biotechnol Adv. 2011), to our knowledge BV transduction efficiency has not been tested in human CD34+ HSPCs or shown in any hematopoietic cell line. Here we show for the first time that BV can be used as a gene delivery vector for primary human CD34+ cells mobilized from healthy donors. We constructed VSV-G pseudotyped BV with a copGFP reporter flanked by 4kb homology arms (HAs) to ITGB2, a locus mutated in leukocyte adhesion deficiency type I (LAD-1) (Fig. A, top). As measured by qPCR, viral DNA was detected in CD34+ cells after transduction at a multiplicity of infection (MOI) of 50, suggesting vector binding and entry in these cells. However, although toxicity was not observed, GFP expression as assessed by flow cytometry was mostly undetectable (less than 0.1%). In contrast, robust (>70%) GFP expression was measured in 293A cells using the same BV vector, suggesting that an inhibitory cellular process was uniquely triggered in primary CD34+ cells following transduction with BV. Recent work has shown that BV can activate cellular innate immune pathways including toll-like receptors (TLRs) (Abe, J Virol 2009) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) (Amalfi S, JVI 2020) resulting in viral clearance and attenuation of gene expression (Ono, JVI 2014). We hypothesized that inhibition of activated cellular innate immune pathways may allow for more efficient BV gene expression in human CD34+ HSPCs. To examine this possibility, we tested over a dozen small molecule inhibitors at multiple doses targeting the major dsDNA sensing innate immune pathways including cGAS-STING (Fig. A, bottom). We found that a 45-minute pre-treatment with the STING inhibitor, H-151, while slightly toxic, enhanced GFP expression several fold, from less than 0.1% to an average of 1.5% in multiple independent donors (Fig. B-C). To improve viability, we also targeted cell death pathways. We tested the pan-caspase inhibitor, zVAD-FMK, which can inhibit both innate activation of gasdermin D (GSDMD), a major dsDNA sensing pathway, as well as apoptotic cell death. We additionally tested the necroptosis inhibitor Nec-1, as necroptosis can be activated in settings of apoptotic inhibition and inflammation. Notably, the combination of both inhibitors with H-151 improved not only cell viability, but also substantially enhanced GFP expression (8%), suggesting a synergistic benefit by inhibiting both innate immune activation and cell death pathways (Fig. D-E). To assess whether BV can efficiently transduce HSPCs with long-term repopulating activity, we pre-stimulated CD34+ cells for 48 hours in culture followed by transduction with BV at an MOI of 25 with our optimized drug cocktail. We examined GFP positivity in both CD34+CD38+ progenitors and CD34+CD38- HSC enriched populations by flow cytometry. After 24 hours, we found an average of 28% GFP+ CD34+CD38- cells and 8% GFP+ CD34+CD38+ progenitors (Fig. F-G). These data suggest that using our optimized approach, BV can target more primitive HSPCs. Collectively, our results lay the groundwork for future studies characterizing innate immune responses to dsDNA viruses in CD34+ cells, and highlight the potential use of BV as a delivery system for homology-directed gene editing in HSPCs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

An intein-mediated split-nCas9 system for base editing in plants

Virus-assisted delivery of the clustered regularly interspaced short palindromic (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. However, the relatively large size of the CRISPR/Cas9 system is challenging to package into viral vectors with confined packaging capacity. To address this technical challenge, we developed a strategy that splits the required CRISPR-Cas9 components across a dual-vector system in which CRISPR-Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by successfully demonstrating split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-SpnCas9 is capable of inducing efficient base editing in plant cells and identified several valid split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.

CRISPR Systems Suitable for Single AAV Vector Delivery

: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas gene editing is a revolutionary technology that can enable the correction of genetic mutations in vivo, providing great promise as a therapeutic intervention for inherited diseases. Adeno-associated viral (AAV) vectors are a potential vehicle for delivering CRISPR/Cas. However, they are restricted by their limited packaging capacity. Identifying smaller Cas orthologs that can be packaged, along with the required guide RNA elements, into a single AAV would be an important optimization for CRISPR/Cas gene editing. Expanding the options of Cas proteins that can be delivered by a single AAV not only increases translational application but also expands the genetic sites that can be targeted for editing. This review considers the benefits and current scope of small Cas protein orthologs that are suitable for gene editing approaches using single AAV vector delivery.

Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer

CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant KrasG12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.

Mucosal priming with a recombinant influenza A virus-vectored vaccine elicits T-cell and antibody responses to HIV-1 in mice

Recombinant influenza A viral (IAV) vectors are potential to stimulate systemic and mucosal immunity, but the packaging capacity is limited and only one or a few epitopes can be carried. Here, we report the generation of a replication-competent IAV vector that carries a full-length HIV-1 p24 gene linked to the 5’-terminal coding region of the neuraminidase segment via a protease cleavage sequence (IAV-p24). IAV-p24 was successfully rescued and stably propagated, and P24 protein was efficiently expressed in infected mammalian cells. In BALB/c mice, IAV-p24 showed attenuated pathogenicity than the parental A/PR/8/34 (H1N1) virus did. An intranasal inoculation with IAV-p24 elicited moderate HIV-specific cell-mediated immune (CMI) responses in the airway and vaginal tracts and in the spleen, and an intranasal boost with a replication-incompetent adenovirus type 2 vector expressing HIV-1 gag gene (Ad2-gag) greatly improved these responses. Importantly, compared to an Ad2-gag prime plus IAV-p24 boost regimen, the IAV-p24 prime plus Ad2-gag boost regimen had a greater efficacy in eliciting HIV-specific CMI responses. P24-specific CD8+ T cells and antibodies were robustly provoked both systemically and in mucosal sites and showed long-term durability, revealing that IAV-p24 may be used as a mucosa-targeted priming vaccine. Our results illustrate that IAV-p24 is able to prime systemic and mucosal immunity against HIV-1 and warrants further evaluation in nonhuman primates. IMPORTANCE An effective HIV-1 vaccine remains elusive despite nearly 40 years of research. CD8+ T cells and protective antibodies may both be desirable for preventing HIV-1 infection in susceptible mucosal sites. Recombinant influenza A virus (IAV) vector has the potential to stimulate these immune responses, but the packaging capacity is extremely limited. Here, we describe a replication-competent IAV vector expressing HIV-1 p24 gene (IAV-p24). Unlike most other IAV vectors that carried one or several antigenic epitopes, IAV-p24 stably expressed the full-length P24 protein which contains multiple epitopes and is highly conserved among all known HIV-1 sequences. Compared to the parental A/PR/8/34 (H1N1) virus, IAV-p24 showed an attenuated pathogenicity in BALB/c mice. When combined with an adenovirus vector expressing HIV-1 gag gene, IAV-p24 was able to prime P24-specific systemic and mucosal immune responses. IAV-p24 as an alternative priming vaccine against HIV-1 warrants further evaluation in nonhuman primates.

Evaluation of the Lipid-binding Properties of Recombinant Dystrophin Spectrin-like Repeat Domains R1-3

Recombinant micro-dystrophin genes are designed to treat Duchenne muscular dystrophy (DMD) by retaining dystrophin domains believed to play key functional roles while fitting the packaging capacity of adeno-associated virus vectors. Domains R1-3 are important for muscle force generation and for association with the sarcolemma, but the nature of this interaction is not fully understood. We measured lipid-binding affinity of 3 peptides containing different spectrin-like repeat modules (R1-3; R1-2; and R1, 2, 22). Lipid-binding affinity was highest with R1-3, suggesting that the complete R1-R3 region could be beneficial and should be considered for inclusion in micro-dystrophin constructs.

Fusion of Large Polypeptides to Human Adenovirus Type 5 Capsid Protein IX Can Compromise Virion Stability and DNA Packaging Capacity

ABSTRACT The human adenovirus (HAdV) protein IX (pIX) is a minor component of the capsid that acts in part to stabilize the hexon-hexon interactions within the mature capsid. Virions lacking pIX have a reduced DNA packaging capacity and exhibit thermal instability. More recently, pIX has been developed as a platform for presentation of large polypeptides, such as fluorescent proteins or large targeting ligands, on the viral capsid. It is not known whether such modifications affect the natural ability of pIX to stabilize the HAdV virion. In this study, we show that addition of large polypeptides to pIX does not alter the natural stability of virions containing sub-wild-type-sized genomes. However, similar virions containing wild-type-sized genomes tend to genetically rearrange, likely due to selective pressure caused by virion instability as a result of compromised pIX function. IMPORTANCE Human adenovirus capsid protein IX (pIX) is involved in stabilizing the virion but has also been developed as a platform for presentation of various polypeptides on the surface of the virion. Whether such modifications affect the ability of pIX to stabilize the virion is unknown. We show that addition of large polypeptides to pIX can reduce both the DNA packaging capacity and the heat stability of the virion, which provides important guidance for the design of pIX-modified vectors.

Engineering a recombinant Herpesvirus saimiri strain by co-culturing transfected and permissive cells

Recombinant herpesviruses can be used as oncolytic therapeutic agents and high packaging capacity vectors for delivering expression cassettes into the cell. Herpesvirus saimiri is a gamma-herpesvirus that normally infects squirrel monkeys but also has a unique ability to infect and immortalize human lymphocytes while allowing them to retain their mature phenotype and functional activity. Recombination of the Herpesvirus saimiri genome in permissive cells is impeded by its resistance to chemical transfection and electroporation. The aim of this study was to develop an effective method for incorporating expression cassettes into the genome of Herpesvirus saimiri without having to transfect a permissive cell culture. Transfected HEK-293T cells expressing glycoproteins of the measles virus vaccine strain were co-cultured with permissive OMK cells infected with Herpesvirus saimiri. Cell fusion and formation of syncytia stimulated recombination between the viral genome and the expression cassette; this allowed us to obtain a recombinant Herpesvirus saimiri variant without chemical transfection in permissive cells. The genetically modified virus expressed a selectable marker and retained its ability to persist in the cell in the latent state; it also caused immortalization of primary lymphoid cells. The proposed approach allows engineering recombinant Herpesvirus saimiri strains carrying a variety of expression cassettes in its genome.

Adeno-Associated Viral Vectors as a Tool for Large Gene Delivery to the Retina

Gene therapy using adeno-associated viral (AAV) vectors currently represents the most promising approach for the treatment of many inherited retinal diseases (IRDs), given AAV’s ability to efficiently deliver therapeutic genes to both photoreceptors and retinal pigment epithelium, and their excellent safety and efficacy profiles in humans. However, one of the main obstacles to widespread AAV application is their limited packaging capacity, which precludes their use from the treatment of IRDs which are caused by mutations in genes whose coding sequence exceeds 5 kb. Therefore, in recent years, considerable effort has been made to identify strategies to increase the transfer capacity of AAV vectors. This review will discuss these new developed strategies, highlighting the advancements as well as the limitations that the field has still to overcome to finally expand the applicability of AAV vectors to IRDs due to mutations in large genes.