Upon viral infection of the liver, CD8+ Tcell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+ Tcells. Surprisingly, we find local initiation of a CD8+ Tcell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1β contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+ DCs, CD8+ Tcells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral Tcell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+ T responses against AAV capsid and transgene product.

Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.

Chimeric antigen receptor Tcells (CAR-T) therapy has shown great potential in tumor treatment. However, many factors impair the efficacy of CAR-T therapy, such as antigenic heterogeneity and loss, limited potency and persistence, poor infiltration capacity, and a suppressive tumor microenvironment. To overcome these obstacles, recent studies have reported a new generation of CAR-T cells expressing cytokines called armored CAR-T, TRUCK-T, or the fourth-generation CAR-T. Here we summarize the strategies of arming CAR-T cells with natural or synthetic cytokine signals to enhance their anti-tumor capacity. Moreover, we summarize the advances in CAR-T cells expressing non-cytokine proteins, such as membrane receptors, antibodies, enzymes, co-stimulatory molecules, and transcriptional factors. Furthermore, we discuss several prospective strategies for armored CAR-T therapy development. Altogether, these ideas may provide new insights for the innovations of the next-generation CAR-T therapy.

Nanofitins are small and hyperthermostable alternative protein scaffolds that display physicochemical properties making them suitable for the development of topical therapeutics, notably for the treatment of pulmonary infectious diseases. Local administration of biologics to the lungs involves a particularly stressful step of nebulization that is poorly tolerated by most antibodies, which limits their application by this delivery route. During the COVID-19 pandemic, we generated anti-SARS-CoV-2 monomeric Nanofitins of high specificity for the spike protein. Hit Nanofitin candidates were identified based on their binding properties with punctual spike mutants and assembled into a linear multimeric construction constituting of four different Nanofitins, allowing the generation of a highly potent anti-SARS-CoV-2 molecule. The therapeutic efficacy of the multimeric assembly was demonstrated both in invitro and invivo models. Interestingly, the neutralization mechanism of the multimeric construction seems to involve a particular conformation switch of the spike trimer. In addition, we reported the stability and the conserved activity of the tetrameric construction after nebulization. This advantageous developability feature for pulmonary administration associated with the ease of assembly, as well as the fast generation process position the Nanofitin technology as a potential therapeutic solution for emerging infectious diseases.

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme's bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.

Genome-wide association studies indicate that allele variants in MIR137, the host gene of microRNA137 (miR137), confer an increased risk of schizophrenia (SCZ). Aberrant expression of miR137 and its targets, many of which regulate synaptic functioning, are also associated with an increased risk of SCZ. Thus, miR137 represents an attractive target aimed at correcting the molecular basis for synaptic dysfunction in individuals with high genetic risk for SCZ. Advancements in nanotechnology utilize lipid nanoparticles (LNPs) to transport and deliver therapeutic RNA. However, there remains a gap in using LNPs to regulate gene and protein expression in the brain. To study the delivery of nucleic acids by LNPs to the brain, we found that LNPs released miR137 cargo and inhibited target transcripts of interest in neuroblastoma cells. Biodistribution of LNPs loaded with firefly luciferase mRNA remained localized to the mouse prefrontal cortex (PFC) injection site without circulating to off-target organs. LNPs encapsulating Cre mRNA preferentially co-expressed in neuronal over microglial or astrocytic cells. Using quantitative proteomics, we found miR137 modulated glutamatergic synaptic protein networks that are commonly dysregulated in SCZ. These studies support engineering the next generation of brain-specific LNPs to deliver RNA therapeutics and improve symptoms of central nervous system disorders.