Targeted nonviral delivery of genome editors in vivo.

Cell-type-specific in vivo delivery of genome editing molecules is the next breakthrough that will drive biological discovery and transform the field of cell and gene therapy. Here, we discuss recent advances in the delivery of CRISPR-Cas genome editors either as preassembled ribonucleoproteins or encoded in mRNA. Both strategies avoid pitfalls of viral vector-mediated delivery and offer advantages including transient editor lifetime and potentially streamlined manufacturing capability that are already proving valuable for clinical use. We review current applications and future opportunities of these emerging delivery approaches that could make genome editing more efficacious and accessible in the future.

CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

Club cells surviving influenza A virus infection induce temporary nonspecific antiviral immunity.

A brief window of antigen-nonspecific protection has been observed after influenza A virus (IAV) infection. Although this temporary immunity has been assumed to be the result of residual nonspecific inflammation, this period of induced immunity has not been fully studied. Because IAV has long been characterized as a cytopathic virus (based on its ability to rapidly lyse most cell types in culture), it has been a forgone conclusion that directly infected cells could not be contributing to this effect. Using a Cre recombinase-expressing IAV, we have previously shown that club cells can survive direct viral infection. We show here not only that these cells can eliminate all traces of the virus and survive but also that they acquire a heightened antiviral response phenotype after surviving. Moreover, we experimentally demonstrate temporary nonspecific viral immunity after IAV infection and show that surviving cells are required for this phenotype. This work characterizes a virally induced modulation of the innate immune response that may represent a new mechanism to prevent viral diseases.

NFX1-123 and human papillomavirus 16E6 increase Notch expression in keratinocytes.

The high-risk human papillomavirus (HR HPV) E6 oncoprotein binds host cell proteins to dysregulate multiple regulatory pathways, including apoptosis and senescence. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and together they posttranscriptionally increase hTERT expression, the catalytic subunit of telomerase. NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity and the avoidance of cellular senescence. Little is known regarding what other transcripts are dependent on or augmented by the association of NFX1-123 with 16E6. Microarray analysis revealed enhanced expression of Notch1 mRNA in 16E6-expressing keratinocytes when NFX1-123 was overexpressed. A moderate increase in Notch1 mRNA was seen with overexpression of NFX1-123 alone, but with 16E6 coexpression the increase in Notch1 was enhanced. The PAM2 motif and R3H protein domains in NFX1-123, which were important for increased hTERT expression, were also important in the augmentation of Notch1 expression by 16E6. These findings identify a second gene coregulated by 16E6 and NFX1-123 and the protein motifs in NFX1-123 that are important for this effect.

In vivo human T cell engineering with enveloped delivery vehicles.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody-antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes.

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

Distinct gene programs underpinning disease tolerance and resistance in influenza virus infection.

When challenged with an invading pathogen, the host-defense response is engaged to eliminate the pathogen (resistance) and to maintain health in the presence of the pathogen (disease tolerance). However, the identification of distinct molecular programs underpinning disease tolerance and resistance remained obscure. We exploited transcriptional and physiological monitoring across 33 mouse strains, during in vivo influenza virus infection, to identify two host-defense gene programs-one is associated with hallmarks of disease tolerance and the other with hallmarks of resistance. Both programs constitute generic responses in multiple mouse and human cell types. Our study describes the organizational principles of these programs and validates Arhgdia as a regulator of disease-tolerance states in epithelial cells. We further reveal that the baseline disease-tolerance state in peritoneal macrophages is associated with the pathophysiological response to injury and infection. Our framework provides a paradigm for the understanding of disease tolerance and resistance at the molecular level.

Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering.

As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus.

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice.

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2.

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.

DNA mismatch repair is required for the host innate response and controls cellular fate after influenza virus infection.

Despite the cytopathic nature of influenza A virus (IAV) replication, we recently reported that a subset of lung epithelial club cells is able to intrinsically clear the virus and survive infection. However, the mechanisms that drive cell survival during a normally lytic infection remained unclear. Using a loss-of-function screening approach, we discovered that the DNA mismatch repair (MMR) pathway is essential for club cell survival of IAV infection. Repair of virally induced oxidative damage by the DNA MMR pathway not only allowed cell survival of infection, but also facilitated host gene transcription, including the expression of antiviral and stress response genes. Enhanced viral suppression of the DNA MMR pathway prevented club cell survival and increased the severity of viral disease in vivo. Altogether, these results identify previously unappreciated roles for DNA MMR as a central modulator of cellular fate and a contributor to the innate antiviral response, which together control influenza viral disease severity.

A Recombinant Antibody-Expressing Influenza Virus Delays Tumor Growth in a Mouse Model.

Influenza A virus (IAV) has shown promise as an oncolytic agent. To improve IAV as an oncolytic virus, we sought to design a transgenic virus expressing an immune checkpoint-inhibiting antibody during the viral life cycle. To test whether it was possible to express an antibody during infection, an influenza virus was constructed encoding the heavy chain of an antibody on the PB1 segment and the light chain of an antibody on the PA segment. This antibody-expressing IAV grows to high titers, and the antibodies secreted from infected cells exhibit comparable functionality with hybridoma-produced antibodies. To enhance the anti-cancer activity of IAV, an influenza virus was engineered to express a single-chain antibody antagonizing the immune checkpoint CTLA4 (IAV-CTLA4). In mice implanted with the aggressive B16-F10 melanoma, intratumoral injection with IAV-CTLA4 delayed the growth of treated tumors, mediated an abscopal effect, and increased overall survival.

Alveolar macrophages are critical for broadly-reactive antibody-mediated protection against influenza A virus in mice.

The aim of candidate universal influenza vaccines is to provide broad protection against influenza A and B viruses. Studies have demonstrated that broadly reactive antibodies require Fc-Fc gamma receptor interactions for optimal protection; however, the innate effector cells responsible for mediating this protection remain largely unknown. Here, we examine the roles of alveolar macrophages, natural killer cells, and neutrophils in antibody-mediated protection. We demonstrate that alveolar macrophages play a dominant role in conferring protection provided by both broadly neutralizing and non-neutralizing antibodies in mice. Our data also reveal the potential mechanisms by which alveolar macrophages mediate protection in vivo, namely antibody-induced inflammation and antibody-dependent cellular phagocytosis. This study highlights the importance of innate effector cells in establishing a broad-spectrum antiviral state, as well as providing a better understanding of how multiple arms of the immune system cooperate to achieve an optimal antiviral response following influenza virus infection or immunization.Broadly reactive antibodies that recognize influenza A virus HA can be protective, but the mechanism is not completely understood. Here, He et al. show that the inflammatory response and phagocytosis mediated by the interaction between protective antibodies and macrophages are essential for protection.

Human papillomavirus 16E6 and NFX1-123 potentiate Notch signaling and differentiation without activating cellular arrest.

High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, but in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21(Waf1/CIP1), or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation.