Hepatitis B virus (HBV) co-opts and interacts with an extensive array of host factors for productive infection. Herein, we developed an HBV reporter virus expressing red fluorescent protein (HBV-RFP) that is suitable for a CRISPR-based genome-wide screen for HBV host-dependency factors. HepG2 cells were transduced with a pooled lentiviral library of single-guide RNA (sgRNA) targeting 19,114 human genes, edited and infected with HBV-RFP. RFP-low cells were sorted using fluorescence-activated cell sorting. The sorted cells were expanded and underwent two additional rounds of infection and sorting to enrich for sgRNA-targeted proviral host factors. By next-generation sequencing and bioinformatic analyses, we identified 63 genes as candidate host proviral factors, including known HBV proviral factors: RXRA, POLL, LDLR and NTCP. Among the novel candidate genes, knock-out of 12 genes significantly decreased HBV replication markers. Validation using siRNA knock-down in primary human hepatocytes confirmed several factors including the monoacylglycerol acyltransferase 2 (MOGAT2) gene as a bona fide HBV pro-viral factor. Further analysis with MGAT2 inhibitors demonstrated that inhibition of MOGAT2 activity impairs HBV transcription and replication. Our study demonstrates the value of the HBV reporter system in identifying previously unrecognized host metabolic factors important for HBV infection, offering a potential avenue for therapeutic development.

Nectin-4 is highly expressed across multiple solid tumor types, and Nectin-4-targeting antibody-drug conjugates (ADCs) have demonstrated promising clinical efficacy. However, the potential of Nectin-4 as a CAR T cell target remains largely unexplored in clinical settings. In this study, we identified multiple Nectin-4 specific antibodies from a fully human phage library and developed corresponding chimeric antigen receptors (CARs). Comprehensive in vitro and in vivo studies revealed that CT293 outperformed other candidates, exhibiting enhanced multifunctionality and superior anti-tumor activity. Notably, CT293, derived from a low-affinity antibody, exhibited no detectable binding to tumor-produced soluble Nectin-4 (sNectin-4) and demonstrated a higher responsiveness threshold compared to its high-affinity counterpart. Clinically, we initiated a first-in-human clinical trial to evaluate the safety profile of CT293 (NCT06724835). Here, we report a case of classic Nectin-4-targeted treatment-associated on-target/off-tumor (OTOT) toxicities following the infusion of CT293 CAR T cells. We provide a detailed characterization of toxicity progression and corresponding clinical management strategies, identifying dermatologic, oromucosal, and gastrointestinal toxicities as the most clinically significant adverse events. Despite Nectin-4 being a valuable drug target, our study underscores the necessity of systematic risk assessment in the development of Nectin-4-targeted cell therapies.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an emerging target in cancer immunotherapy, recognized for its consistent and elevated expression across several epithelial tumors, including triple-negative breast cancer (TNBC). TNBC is an aggressive and difficult-to-treat cancer, with limited effective therapeutic options currently available. Therapeutic approaches centered on targeting ROR1 have therefore become increasingly popular, with ROR1 chimeric antigen receptor (CAR) T cells currently in clinical trials to treat TNBC patients. While ROR1-targeting therapies have shown promising preclinical results, single arm treatment has often shown low efficacy as well as off-target toxicity. Natural killer (NK) cell-based immunotherapies, such as antibody-dependent cell cytotoxicity-inducing monoclonal antibodies and CAR NK cells, have also been shown to induce cancer cell cytotoxicity; however, with less toxicity compared with CAR T cells. Here, we developed and characterized a phage-derived single-chain fragment variable (scFv) against a highly specific ROR1 region and generated scFv-derived chimeric monoclonal antibodies and anti-ROR1-CAR NK cells, which show anti-cancer efficacy against TNBC cells. Additionally, we found TGF-β inhibition using either small-molecule inhibitors or CRISPR-Cas9-edited NK cells could further enhance ROR1-targeting therapy persistence and efficacy in controlling TNBC tumor growth.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe genetic disorder caused by bi-allelic null mutations in the COL7A1 gene. Among various therapeutic genome editing (GE) strategies, adenine base editors (ABEs) have demonstrated particular efficacy in nonsense mutations. Previous studies have predominantly focused on RDEB fibroblasts, which produce significantly less type VII collagen (C7) than keratinocytes. This study evaluates the efficacy and safety of ABE8e, via mRNA electroporation, to correct three different COL7A1 nonsense mutations (one homozygous and two heterozygous [patients 1, 2, and 3]), and a cryptic splice site mutation (patient 4) in primary cells from RDEB patients. Sanger and next-generation sequencing revealed high efficiency (>90%) at the intended mutation-causing nucleotides. Bystander effects were observed in two of the four loci analyzed. Deeper off-target study was performed in the first two patients. A safe off-target profile was observed in the homozygous patient (P1), while off-target effects were identified in the heterozygous patient (P2). The restoration of C7 expression in treated cells was consistent with genotypic correction. Additionally, functional assessments, including transplantation of skin equivalents formed by both base-edited keratinocytes and fibroblasts, demonstrated sustained in vivo skin regeneration and proper C7 deposition at the basement membrane. ABE8e-mediated base editing shows high efficacy and safety correcting COL7A1 mutations in the two cell types, broadening the scope of this GE therapeutic modality for RDEB treatment.

Defects in mitochondrial energy metabolism in injured tubular epithelial cells (TECs) are a well-recognized hallmark of kidney injury pathogenesis; however, the key target leading to this defect during the acute kidney injury (AKI)-to-chronic kidney disease (CKD) transition remains elusive. Here, we found that during the AKI-to-CKD transition, the increased WW domain containing E3 ubiquitin protein ligase 2 (WWP2) was shuttled to the mitochondria and disabled TEC mitochondrial energy metabolism by ubiquitinating and degrading complex II subunit succinate dehydrogenase complex subunit C (SDHC), leading to oxidative phosphorylation (OXPHOS) disability and aggravated TEC maladaptive repair. Preemptive and late depletion of Wwp2 both ameliorated unilateral ischemia-reperfusion (UIR) injury-induced AKI-to-CKD transition, and tubular-specific Wwp2 depletion resulted in the same protective phenotype. Furthermore, Sdhc knockdown abolished the protective effects of Wwp2 deletion in UIR mice. Conversely, SDHC overexpression attenuated OXPHOS impairment and TEC injury following WWP2 overexpression. Finally, we leveraged high-throughput virtual screening, enzyme activity assays, and binding affinity assays to identify two candidate WWP2 inhibitors. Both inhibitors significantly improved TEC maladaptive repair and deferred the AKI-to-CKD transition. Overall, we identified WWP2 as a critical regulator of mitochondrial OXPHOS integrity in maladaptive repairing TECs and identified two WWP2 inhibitors as potential drug candidates for interrupting the AKI-to-CKD transition.

Renal fibrosis is a common histological feature of chronic kidney disease. The role of phosphodiesterase 1A (PDE1A), a crucial molecule regulating cyclic guanosine monophosphate (cGMP) signaling in tubular epithelial cells, in renal fibrosis is unknown. We found that the mRNA of PDE1A were elevated in the urinary exosomes from patients with rapidly progressing IgA nephropathy. The PDE1A protein in renal tubular epithelial cells increased as renal function decline in patients with IgA nephropathy or diabetic kidney disease. In mouse models of unilateral ureter obstruction (UUO) and chronic folic acid nephropathy (FAN), renal interstitial fibrosis was significantly alleviated in tubular epithelial cell-specific Pde1a knockout (Pde1a-cKO) mice, or after treatment with ITI-214, a PDE1 inhibitor. In HK-2 cells, transforming growth factor-β1 (TGF-β1) incubation induced PDE1A expression, which was prevented by pretreatment with ITI-214 or Pde1a siRNA. Overexpression of PDE1A exacerbated TGF-β1-induced matrix protein production. Inhibition of PDE1A reversed the TGF-β1-induced decrease in cGMP levels and Protein kinase G (PKG) signaling, without affecting cyclic adenosine monophosphate (cAMP) levels. We demonstrated that PKG directly interacts with Thr41of β-catenin, leading to an increase in phosphorylated β-catenin (Ser33/37/Thr41). PKG activation and inactivation of β-catenin were verified in UUO and FAN model in Pde1a-cKO mice or ITI-214 treated mice. Furthermore, vericiguat, a soluble guanylate cyclase(sGC) agonist, induced cGMP signaling and mitigated renal fibrosis in UUO mice. These findings suggest that PDE1A may play a key role in renal fibrosis. Both PDE1A inhibition or gene knockout, and sGC activation can prevent the progression of renal fibrosis.

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.

Pain and inflammation are the most common symptoms experienced by patients with acute rheumatoid arthritis (RA). Immunomodulatory signals released by nociceptive nerves play an important role in various infectious and inflammatory diseases. In this study, we found that ablation of nociceptive nerves exacerbated joint inflammation in an antigen-induced arthritis model. Our findings revealed that immune complex could independently and directly activate nociceptive nerves in the synovium, initiating pain signals that subsequently modulate inflammation through the neuropeptide calcitonin gene-related peptide (CGRP). Specifically, CGRP bound to the receptor activity-modifying protein 1 (Ramp1) and calcitonin receptor-like receptor (Calcrl) complex on CX3CR1 synovial macrophages, suppressing the expression of inflammatory factors and chemokines while inhibiting the recruitment of immune cells. However, prolonged activation of nociceptive nerves led to the downregulation of Calcrl in synovial macrophages, reducing their sensitivity to CGRP. This diminished sensitivity implied that the excessive release of pain-related transmitters failed to suppress inflammation effectively. To address this, we overexpressed Calcrl in CX3CR1 synovial macrophages by transfecting them with recombinant adeno-associated virus (rAAV), demonstrating that this approach alleviates joint inflammation without intensifying pain. These findings highlight the therapeutic potential of targeting neuroimmune interactions to manage RA.

Hereditary Spastic Paraplegia type 4 is characterized by gait impairments, progressive spasticity and weakness of the lower limbs, resulting from degeneration of the corticospinal tracts. The disease is caused by mutations of the SPAST gene, which encodes a major isoform of spastin called M87 and a minor isoform called M1. Owing to its N-terminal hydrophobic domain not shared by M87, M1 is the isoform that becomes toxic when mutated. Loss-of-function of either M1 or M87 or both may also play a role in the disease, sensitizing corticospinal motor neurons to the toxicity of mutant M1. Here we pursued silence-and-replace gene therapy, which addresses both gain-of-toxicity and loss-of-function components of the disease. We generated an adeno-associated serotype 9 viral vector containing micro-RNA to stop the expression from the endogenous SPAST gene and cDNA to express healthy human M1 and M87. The vector was introduced by intracerebroventricular injections into newborn pups of SPAST-C448Y, a mouse model of the disease that expresses human mutant spastin and displays adult-onset corticospinal degeneration and gait defects. The treatment successfully replaced both isoforms of endogenous spastin with healthy spastin, at physiological levels, and prevented the onset and progression of corticospinal degeneration and gait defects.

Triple-negative breast cancer (TNBC) is a form of breast cancer clarified by low expression of estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). For this reason, therapeutics aimed at targeting these receptors are ineffective in cases of TNBC, which leads to a poorer prognosis. Consequently, there is a need for novel therapeutics at targeting this subtype. CCAAT/enhancer-binding protein beta (C/EBPβ) is a leucine zipper transcription factor with a traditional function in mammary gland development and macrophage differentiation. In tumors, C/EBPβ is associated with metastatic and chemoresistant forms of breast cancer. Previous efforts at targeting this transcription factor in the tumor have been hampered by off-target effects and low penetrance into the intratumoral space. Furthermore, studies into C/EBPβ knockdown in vitro have been mixed, owing in part to two distinct isoforms that are differentially expressed in healthy and cancerous tissues. Given that C/EBPβ's function is closely tied to hypoxia factors such as HIF-1α, we hypothesized that the hypoxic intratumoral space may be driving specific isoform development, and consequently the pro-metastatic phenotype observed clinically. To this end, we have developed an aptamer-siRNA conjugate containing a transferrin receptor 1 (TfR1) aptamer (a receptor activated under hypoxic conditions) linked to a C/EBPβ siRNA. We have measured C/EBPβ's suppression of metastasis in traditional cell culture under hypoxic conditions and in vivo. These results point toward a novel approach to C/EBPβ's contradictory role as a driver and mediator of metastasis, and a potential therapeutic for its treatment.

mRNA has revolutionized vaccine development, demonstrating high efficacy and safety in COVID-19 vaccines, and is now being explored for broader therapeutic applications. However, while vaccines rely on widespread antigen expression, many therapeutic strategies-particularly in oncology-require precise, cell-selective gene expression. Here, we present the selective modified RNA translation system (SMRTS), a versatile, engineered mRNA system that enables targeted gene expression in specific cell populations. As a proof of concept, we developed cancer-specific variants, bcSMRTS and ccSMRTS, for breast and colon cancer, respectively. Systemic delivery of lipid nanoparticle (LNP)-encapsulated SMRTS constructs yielded a 114-fold and 141-fold increase in tumor-specific expression in 4T1 and MC-38 models, respectively, while reducing off-target expression by over 380-fold. Therapeutic deployment of Pten ccSMRTS suppressed tumor growth by 45%, and combination with modRNA-derived anti-checkpoint inhibitor antibodies (modRNabs) resulted in up to 93% tumor inhibition. Beyond oncology, SMRTS introduces a novel mRNA tool, providing a versatile system for cell-selective gene expression. By expanding the mRNA therapeutics toolbox, SMRTS paves the way for precise mRNA-based interventions across a wide range of disease settings.

Although an emerging body of evidence suggests that abnormal forms of tau are present and contribute to Huntington's disease - a genetic neurodegenerative disorder primarily characterized by the aggregation of the mutant huntingtin protein affecting cognitive, motor, and psychiatric function - it is not clear to what extent this is relevant to the disease phenotype, and hence future treatments. We therefore generated a novel murine model by crossing heterozygous zQ175 knock-in HD mice with homozygous tau knockout mice. Tau deletion exacerbated both motor and cognitive deficits in zQ175/mTKO mice which was accompanied by increased mHtt aggregation and alterations in microtubule dynamics, including dysregulated expression of microtubule-associated proteins, aberrant perinuclear β-tubulin accumulation, and microtubule destabilization. Combined, our findings unveil a previously unrecognized protective role of non-hyperphosphorylated tau in maintaining cytoskeletal homeostasis in HD and highlight a functional overlap between tau and huntingtin in regulating aggregate dynamics and cytoskeletal integrity. Our findings complement previous reports suggesting that reducing tau levels could mitigate disease pathology in HD mouse models.