Molecular Therapy最新文献

Dorsal root ganglion (DRG) toxicity has been consistently reported as a potential safety concern after delivery of adeno-associated viruses (AAVs) containing gene replacement vectors but has yet to be reported for RNAi-based vectors. Here, we report DRG toxicity after AAV intra-CSF delivery of an RNAi expression construct-artificial miRNA targeting superoxide dismutase 1 (SOD1)-in nonhuman primates (NHPs) and provide evidence this can be recapitulated within mice. Histopathology evaluation showed that NHPs and mice develop DRG toxicity after AAV delivery, including DRG neuron degeneration and necrosis, and nerve fiber degeneration that were associated with increases in cerebrospinal fluid (CSF) and serum phosphorylated neurofilament heavy chain (pNF-H). RNA-seq analysis of DRGs showed that dysregulated pathways were preserved between NHPs and mice, including increases in innate/adaptive immune responses, and decreases in mitochondrial- and neuronal-related genes following AAV treatment. Finally, endogenous miR-21-5p was upregulated in DRGs of AAV-treated NHPs and mice. Increases in miR-21-5p were also identified within the CSF of NHPs, which significantly correlated with pNF-H, implicating miR-21-5p as a potential biomarker of DRG toxicity in conjunction with other molecular analytes. This work highlights the importance of assessing safety concerns related to DRG toxicity when developing RNAi-based AAV vectors for therapeutic purposes.

Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. MiR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector influenced glioma development resulting in beneficial anti-tumor outcomes in GB-bearing mice.

Despite a dramatic increase in ischemic stroke incidence worldwide, effective therapies for attenuating sequelae of cerebral infarction are lacking. This study investigates the use of human mesenchymal stem cells (hMSCs) induced toward glia-like cells (ghMSCs) to ameliorate chronic sequelae resulting from cerebral infarction. Transcriptome analysis demonstrated that ghMSCs exhibited astrocytic characteristics, and assessments conducted ex vivo using organotypic brain slice cultures demonstrated that ghMSCs exhibited superior neuroregenerative and neuroprotective activity against ischemic damage compared to hMSCs. The observed beneficial effects of ghMSCs were diminished by pre-treatment with a CXCR2 antagonist, indicating a direct role for CXCR2 signaling. Studies conducted in rats subjected to cerebral infarction demonstrated that ghMSCs restored neurobehavioral functions and reduced chronic brain infarction in a dose-dependent manner when transplanted at the subacute-to-chronic phase. These beneficial impacts were also inhibited by a CXCR2 antagonist. Molecular analyses confirmed that increased neuroplasticity contributed to ghMSCs' neuroregenerative effects. These data indicate that ghMSCs hold promise for treating refractory sequelae resulting from cerebral infarction by enhancing neuroplasticity and identify CXCR2 signaling as an important mediator of ghMSCs' mechanism of action.

Lipoprotein(a), or Lp(a), is encoded by the LPA gene and is a causal genetic risk factor for cardiovascular disease. Individuals with high Lp(a) are at risk for cardiovascular morbidity and are refractory to standard lipid-lowering agents. Lp(a)-lowering therapies currently in clinical development require repetitive dosing, while a gene editing approach presents an opportunity for a single-dose treatment. In this study, mRNAs encoding Transcription Activator-Like Effector Nucleases (TALENs) were designed to target human LPA for gene disruption and permanent Lp(a) reduction. TALEN mRNAs were screened in vitro and found to cause on-target gene editing and target protein reduction with minimal off-target editing. TALEN mRNAs were then encapsulated with LUNAR®, a proprietary lipid nanoparticle (LNP), and administered to transgenic mice that expressed a human LPA transgene. A single dose of TALEN mRNA-LNPs reduced plasma Lp(a) levels in mice by over 80%, which was sustained for at least 5 weeks. Moreover, both standard and long-read next generation sequencing confirmed the presence of gene-inactivating deletions at LPA transgene loci. Overall, this study serves as a proof-of-concept for using TALEN-mediated gene editing to disrupt LPA in vivo, paving the way for the development of a feasible gene editing therapy for patients with high Lp(a).

Long-read RNA sequencing (RNA-seq) is emerging as a powerful and versatile technology for studying human transcriptomes. By enabling the end-to-end sequencing of full-length transcripts, long-read RNA-seq opens up avenues for investigating various RNA species and features that cannot be reliably interrogated by standard short-read RNA-seq methods. In this review, we present an overview of long-read RNA-seq, delineating its strengths over short-read RNA-seq, as well as summarizing recent advances in experimental and computational approaches to boost the power of long-read based transcriptomics. We describe a wide range of applications of long-read RNA-seq, and highlight its expanding role as a foundational technology for exploring transcriptome variations in human diseases.

Immunostimulatory cytokines and immune checkpoint inhibitors hold promise as cancer therapeutics; however, their use is often limited by reduced efficacy and significant toxicity. In this study, we developed small-format immunocytokines (ICKs) based on interleukin-12 (IL-12) and blocking nanobodies targeting mouse and human PD-1 and PD-L1. Both PD-1 and PD-L1-targeted ICKs demonstrated similar in vitro performance, significantly increasing IL-12 tethering to immune cells and enhancing T cell cytotoxic activity compared to IL-12 alone. Antitumor efficacy of ICKs was evaluated by intratumoral delivery using self-amplifying RNA-based vectors or as recombinant proteins in mice. Despite effective PD-L1-mediated tumor anchoring and promising in vitro results, IL-12 antitumor activity was significantly enhanced only when specific targeting to intratumoral T cells was achieved via anti-PD-1 nanobody. This effect was also observed when the PD-1 specific ICK was delivered by electroporation of a DNA/RNA layered vector. Our findings suggest that targeting the appropriate type of cell within the tumor microenvironment could outperform tumor-anchoring strategies in the context of IL-12 therapy. Human versions of these ICKs were also developed, which showed to be active in human immune cells, opening an opportunity for clinical translation.

CD19 CAR-T therapy has achieved remarkable responses in relapsed/refractory non-Hodgkin lymphoma (NHL). However, challenges persist, with refractory responses or relapses after CAR-T administration linked to CD19 loss or downregulation. Given the co-expression of CD19 and BCMA in NHL, we hypothesized that dual-targeting could enhance long-term efficacy. We optimized different dual-targeting approaches, including co-transduction of two lentiviral vectors, bicistronic, tandem, loop and pool strategies, based on our academic anti-CD19 (ARI0001) and anti-BCMA (ARI0002h) CAR-T cells. Comparison with anti-CD19/CD20 or anti-CD19/CD22 dual-targeting was also performed. We demonstrate that anti-CD19/BCMA CAR-T cells can be effectively generated through co-transduction of two lentiviral vectors after optimization to minimize competition for cellular resources. Co-transduced T cells, named ARI0003, effectively targeted NHL tumor cells with high avidity, outperforming anti-CD19 CAR-T cells and other dual-targeting approaches both in vitro and in vivo, particularly in low CD19 antigen density models. ARI0003 maintained effectiveness post-CD19 CAR-T treatment in xenograft models and in spheroids from relapsed CART-treated patients. ARI0003 CAR-T cells were effectively manufactured under Good Manufacturing Practice conditions, with reduced risk of genotoxicity compared to other dual-targeting approaches. A first-in-human phase I clinical trial (CARTD-BG-01, NCT06097455) has been initiated to evaluate the safety and efficacy of ARI0003 in NHL.

Monoclonal antibodies are an important class of biologics with over 160 FDA/EU approved drugs. A significant bottleneck to global accessibility of recombinant monoclonal antibodies stems from complexities related to their production, storage, and distribution. Recently, gene-encoded approaches such as mRNA, DNA or viral delivery have gained popularity, but ensuring biologically relevant levels of antibody expression in the host remains a critical issue. Using a synthetic DNA platform, we investigate the role of antibody structure and sequence toward in vivo expression. SARS-COV2 antibody 2196 was recently engineered as a DNA-encoded monoclonal antibody (DMAb-2196). Utilizing an immunoglobulin heavy and light chain "chain-swap" methodology, we interrogate features of DMAb-2196 that can modulate in vivo expression through rational design and structural modeling. Comparing these results to natural variation of antibody sequences resulted in development of an antibody frequency score that aids in the prediction of expression-improving mutations by leveraging antibody repertoire datasets. We demonstrate that a single amino acid mutation identified through this score increases in vivo expression up to 2-fold and that combinations of mutations can also enhance expression. This analysis has led to a generalized pipeline that can unlock the potential for in vivo delivery of therapeutic antibodies across many indications.

Longitudinal, non-invasive, in vivo monitoring of therapeutic gene expression is an unmet need for gene therapy (GT). Positron Emission Tomography (PET) radiotracers designed to bind to therapeutic proteins may provide a sensitive imaging platform to guide treatment response and dose optimization in GT. Herein, we evaluated a novel PET tracer ([¹⁸F]AGAL) for targeting alpha galactosidase A (α-GalA), an enzyme deficient in Fabry disease. Gla knockout mice were subjected to either GT with an adeno-associated virus encoding the human a-Gal A (AAV_GLA) or recombinant α-GalA for enzyme replacement studies. PET imaging, ex vivo autoradiography, biochemical analyses and radiation dosimetry were performed. [¹⁸F]AGAL exhibited pH-dependent binding to a-GalA, suggesting recognition of the active enzyme residing within the acidified lysosomes. Imaging studies in the Fabry mouse model showed quick renal clearance with high radioactive uptake in the heart at 6 weeks that was sustained for 26 weeks after a single administration of AAV_GLA, indicating effective and durable transgene expression from GT. Good concordance was achieved between in vivo PET imaging and ex vivo quantification of α-GalA levels in biofluids and tissues. Biodistribution and dosimetry in non-human primate showed acceptable radiation exposure for multiple injections demonstrating its potential for translation to clinical trial use.

CRISPR/Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein (RNP) delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated sgRNAs to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein:sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing knockout and activation efficiencies. We demonstrate high efficiencies of trimodal genetic engineering in primary human T cells while preserving basic T cell health and functionality. This study highlights the versatility and potential of complex genetic engineering using multiple CRISPR/Cas systems in a simple, one-step process yielding transient transcriptome modulation and permanent DNA changes. We believe such elaborate engineering can be implemented in regenerative medicine and therapies to facilitate more sophisticated treatments.