Functional analysis of tRNA-derived small translational regulation.

Transfer RNAs (tRNAs) are short non-coding RNA molecules that play a crucial role in protein synthesis by carrying amino acids to ribosomes during translation. tRNAs are highly conserved and abundant across species, with each type categorized based on its anticodon sequence. Although traditionally viewed as essential for protein synthesis, tRNAs have been found to have additional roles in cell proliferation, tumor metastasis, and neuronal homeostasis.

Operationalising a Haemophilia Gene Editing Lexicon for Practical Use.

Introduction: Gene editing therapies offer the possibility of substantial improvement in treatment and quality of life for people with haemophilia (PWH) in a landscape of dynamic therapeutic advancement. Developing a common and understandable language to discuss gene editing will be essential to ensure these treatments can be deployed in a safe and effective manner with fully informed and shared decision-making between healthcare professionals (HCPs) and PWH. A lexicon explaining and clarifying key concepts is one potential tool to address these aims.

A novel gene editing lexicon strategy for the haemophilia community: Research plan for development and preliminary results.

Introduction: Despite the progress in gene editing platforms like CRISPR/Cas9 with the potential to transform the standard of care for haemophilia, the language used to explain and discuss gene editing is not aligned across the haemophilia community. Here, we present the objective and rationale for developing a clear, consistent, and globally aligned gene editing lexicon to address these communication gaps.

Methods: Effectively communicating complex gene editing concepts requires a clear and consistent vocabulary.

AAV-mediated genome editing is influenced by the formation of R-loops.

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue.

AAV-mediated genome editing is influenced by the formation of R-loops.

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue.

Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering.

Selection of viral capsids and promoters affects the efficacy of rescue of -deficient cochlea.

Adeno-associated virus (AAV)-mediated gene transfer has shown promise in rescuing mouse models of genetic hearing loss, but how viral capsid and promoter selection affects efficacy is poorly characterized. Here, we tested combinations of AAVs and promoters to deliver , mutations in which are associated with hearing loss in humans. mice display severe cochlear hair cell degeneration, loss of auditory brainstem responses, and delayed loss of spiral ganglion neurons.

A strategy for high antibody expression with low anti-drug antibodies using AAV9 vectors.

Introduction: Use of adeno-associated virus (AAV) vectors is complicated by host immune responses that can limit transgene expression. Recent clinical trials using AAV vectors to deliver HIV broadly neutralizing antibodies (bNAbs) by intramuscular administration resulted in poor expression with anti-drug antibodies (ADA) responses against the bNAb.

Methods: Here we compared the expression of, and ADA responses against, an anti-SIV antibody ITS01 when delivered by five different AAV capsids.

Aptamer-programmable adeno-associated viral vectors as a novel platform for cell-specific gene transfer.

Adeno-associated viruses (AAVs) are commonly used for gene therapy. Nevertheless, the wide tropism that characterizes these vectors limits specific targeting to a particular cell type or tissue. Here, we developed new chemically modified AAV vectors (Nε-AAVs) displaying a single site substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry.

Selection of rAAV vectors that cross the human blood-brain barrier and target the central nervous system using a transwell model.

A limitation for recombinant adeno-associated virus (rAAV)-mediated gene transfer into the central nervous system (CNS) is the low penetration of vectors across the human blood-brain barrier (BBB). High doses of intravenously delivered vector are required to reach the CNS, which has resulted in varying adverse effects. Moreover, selective transduction of various cell types might be important depending on the disorder being treated.

Evaluating the state of the science for adeno-associated virus integration: An integrated perspective.

On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice.

Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice.

Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold.

Selective Microvascular Tissue Transfection Using Minicircle DNA for Systemic Delivery of Human Coagulation Factor IX in a Rat Model Using a Therapeutic Flap.

Background: Gene therapy is a promising treatment for protein deficiency disorders such as hemophilia B. However, low tissue selectivity and efficacy are limitations of systemic vector delivery. The authors hypothesized that selective transfection of rat superficial inferior epigastric artery flaps could provide systemic delivery of coagulation factor IX, preventing the need for systemic vector administration.

The 3'tsRNAs are aminoacylated: Implications for their biogenesis.

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG3´tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice.

RNA structure probing reveals the structural basis of Dicer binding and cleavage.

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e.

Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex.

Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by ∼6- to 9-fold.

Evaluating the Genomic Parameters Governing rAAV-Mediated Homologous Recombination.

Recombinant adeno-associated virus (rAAV) vectors have the unique ability to promote targeted integration of transgenes via homologous recombination at specified genomic sites, reaching frequencies of 0.1%-1%. We studied genomic parameters that influence targeting efficiencies on a large scale.

Promoterless, Nuclease-Free Genome Editing Confers a Growth Advantage for Corrected Hepatocytes in Mice With Methylmalonic Acidemia.

Background And Aims: Adeno-associated viral (AAV) gene therapy has shown great promise as an alternative treatment for metabolic disorders managed using liver transplantation, but remains limited by transgene loss and genotoxicity. Our study aims to test an AAV vector with a promoterless integrating cassette, designed to provide sustained hepatic transgene expression and reduced toxicity in comparison to canonical AAV therapy.

Approach And Results: Our AAV vector was designed to insert a methylmalonyl-CoA mutase (MMUT) transgene into the 3' end of the albumin locus and tested in mouse models of methylmalonic acidemia (MMA).

Transfer RNA-Derived Small RNAs: Another Layer of Gene Regulation and Novel Targets for Disease Therapeutics.

Decades after identification as essential for protein synthesis, transfer RNAs (tRNAs) have been implicated in various cellular processes beyond translation. tRNA-derived small RNAs (tsRNAs), referred to as tRNA-derived fragments (tRFs) or tRNA-derived, stress-induced RNAs (tiRNAs), are produced by cleavage at different sites from mature or pre-tRNAs. They are classified into six major types representing potentially thousands of unique sequences and have been implicated to play a wide variety of regulatory roles in maintaining normal homeostasis, cancer cell viability, tumorigenesis, ribosome biogenesis, chromatin remodeling, translational regulation, intergenerational inheritance, retrotransposon regulation, and viral replication.