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Amendola, M.

Publications and source records attributed to Amendola, M..

8 recordsLinked to original sources
bioRxiv2026

A Single Lipid Nanoparticle Formulation Enables Delivery of Diverse RNA Therapeutics to Human Muscle Models of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a lethal neuromuscular disorder caused by the absence of dystrophin, for which no curative treatment is available. RNA-based approaches have shown promising results; however, their evaluation is hindered by the lack of robust and rapid delivery methods for differentiated human muscle cells, which represent the most physiologically relevant in vitro models for assessing therapeutic strategies. Here, we establish a versatile lipid nanoparticle platform enabling efficient delivery of diverse RNA therapeutics across a range of human muscle models, including myotubes, induced pluripotent stem cell-derived myotubes, myoblasts, cardiomyocytes, and 3D engineered skeletal muscle tissues. Remarkably, a single commercially available lipid nanoparticle formulation supports delivery of cargos spanning more than 300-fold in size, from short antisense oligonucleotides ([~]20 nt) to complex CRISPR-based editors (up to [~]6.7 kb), including Cas9 nucleases, adenine base editors, and CRISPRa systems. This enables efficient gene correction and transcriptional modulation, resulting in dystrophin restoration or compensatory utrophin upregulation in relevant Duchenne muscular dystrophy models. Together, our results establish a single lipid nanoparticle formulation as a versatile platform for RNA delivery in human muscle systems and provide a practical framework for the rapid preclinical assessment of emerging therapies for Duchenne muscular dystrophy and other neuromuscular disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/741396v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a18d71org.highwire.dtl.DTLVardef@4d0faforg.highwire.dtl.DTLVardef@140b078org.highwire.dtl.DTLVardef@1a235_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Leclerc, D. (2026) https://BioRender.com/90h0fme

molecular biology↗
bioRxiv2026

CRISPR-Mediated Linearization of IDLV Donor Enables Precise Targeted Integration in Human Hematopoietic Stem Cells

Ex vivo genome editing of human hematopoietic stem and progenitor cells (HSPCs) requires targeted integration strategies that support large therapeutic DNA payloads while preserving stem cell fitness. Although CRISPR/Cas9-mediated homology-directed repair using AAV donors is effective, it is constrained by limited cargo capacity and adverse effects on long-term HSPCs function. Integrase-defective lentiviral vectors (IDLVs) offer an alternative donor platform, yet their precise and controlled genomic integration remains inefficient. Here, we describe TILV (Targeted Integration of Lentiviral Vector), a CRISPR-assisted knock-in strategy that exploits Cas9-mediated linearization of episomal IDLV DNA to expose a single homology arm and engage homology-mediated end-joining repair pathways. TILV enables precise, directional and seamless integration of transgenes in multiple loci, enabling constitutive or physiological expression. Using single-cell clonal analyses and targeted long-read sequencing, we define the molecular features of TILV-mediated integration and demonstrate preferential use of CRISPR-linearized episomal substrates. TILV supports accurate insertion of large therapeutic transgenes, without compromising HSPC viability or multilineage potential. We further show that transient modulation of DNA repair pathway, in combination with extended homology arms, enhances integration efficiency and junctional precision. Importantly, optimized TILV enables targeted integration in phenotypically defined long-term HSPCs, highlighting its potential for scalable and durable gene therapy.

molecular biology↗
bioRxiv2025

Human liver organoids uncover ductular reaction in Wolman disease and inform gene therapy design

Wolman disease (WD), the severe infantile form of lysosomal acid lipase deficiency, is a rare metabolic disorder caused by inactivating mutations in the LIPA gene. Although WD is characterized by profound hepatic dysfunction, experimental human systems capable of modelling multicellular liver pathology and supporting therapeutic testing remain limited. Here, we generated an isogenic human model of WD by introducing LIPA loss-of-function mutations into induced pluripotent stem cells and differentiating them into multicellular human liver organoids (HLO). LIPA-deficient HLO preserved hepatic lineage specification while recapitulating key biochemical and cellular features of WD, including loss of LIPA activity, lysosomal expansion, lipid accumulation, and activation of inflammatory and fibrogenic programs. Single-cell RNA sequencing resolved cell-type-specific disease states across hepatocyte-, stromal-, and biliary-like populations, revealing the emergence of a reactive biliary program consistent with ductular reaction, a complex tissue response associated with chronic liver injury. Importantly, this reactive biliary phenotype was supported by targeted gene-expression analysis in WD liver organoids and independently validated in liver tissue from mouse models and WD patients. Isolated LIPA-deficient cholangiocyte organoids failed to reproduce the DR-associated program, indicating that this response depends on multicellular interactions within the hepatic microenvironment rather than on biliary cell-autonomous dysfunction alone. Consistently, hepatocyte-directed AAV-mediated restoration of LIPA expression attenuated metabolic stress, inflammatory and fibrogenic programs, and suppressed ductular reaction both in organoids and in vivo. Together, these findings establish multicellular human liver organoids as a physiologically relevant platform for modelling emergent tissue-level responses in WD and for evaluating therapeutic rescue strategies in a human context.

pathology↗
bioRxiv2025

Advanced pipeline for CRISPR/Cas9 offtargets detection in Guide-seq and related integration-based assays

We developed GENETHOFF, a flexible single-command versatile Snakemake workflow designed for the comprehensive analysis of CRISPR/Cas9 related OFF-targets genomic positions from GUIDE-Seq derived protocols. It efficiently processes multiplexed libraries from different organisms, PCR orientations, and Cas nucleases with varying PAM specificities in a single run, all based on a simple user-specified datasheet containing sample metadata.

bioinformatics↗
bioRxiv2024

Rescue of lysosomal acid lipase deficiency in mice by rAAV8 liver gene transfer.

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive disorder caused by mutations in the LIPA gene, which results in lipid accumulation leading to multi-organ failure. If left untreated, the severe form of LAL-D results in premature death within the first year of life due to failure to thrive and hepatic insufficiency. Enzyme replacement therapy is the only available supportive treatment consisting in weekly systemic injections of recombinant LAL protein. Here, we characterized a novel Lipa-/- mouse model and developed a curative gene therapy treatment based on the in vivo administration of recombinant (r)AAV8 vector encoding the human LIPA transgene under the control of a hepatocyte-specific promoter. We defined the minimal rAAV8 dose required to rescue disease lethality and to correct cholesterol and triglyceride accumulation in multiple organs and blood. Finally, using liver transcriptomic and biochemical analysis, we showed mitochondrial impairment in Lipa-/- mice and its recovery by gene therapy. Overall, our in vivo gene therapy strategy achieves a stable long-term LAL expression sufficient to correct the disease phenotype in the Lipa-/-mouse model and offers a new therapeutic option for LAL-D patients. One Sentence SummaryWeve developed a liver-targeted gene therapy using recombinant AAV8 to effectively cure Lysosomal acid lipase deficiency by correcting lipid accumulation and by normalizing gene expression pattern and mitochondrial function in Lipa-/- mouse model.

molecular biology↗
bioRxiv2023

CRISPR-Cas9 mediated endogenous utrophin upregulation improves Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulation of utrophin (UTRN), a dystrophin paralogue, is a promising therapeutic avenue. Here, we present a CRISPR-Cas9-mediated strategy to increase utrophin expression by disrupting microRNA (miR) binding sites (BS). Using a Cas9/gRNA ribonucleoprotein (RNP) complex we disrupted several miR BS in DMD myoblasts and selected the Let-7c BS has crucial for UTRN repression. Interestingly, Cas9/gRNA indels were as efficient as the complete removal of Let-7c BS in upregulating UTRN expression, without any major off-targets. In three-dimensional human DMD cultures, Cas9/gRNA-mediated editing resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, Let-7c BS disruption in mdx animals by systemic rAAVs mediated delivery of Cas9 and gRNA resulted in utrophin upregulation and amelioration of the muscle histopathological phenotype. These findings provide the foundations for a universal (mutation-independent) gene editing therapeutic strategy for DMD. One Sentence SummaryCRISPR-Cas9 has the potential to upregulate utrophin to treat all DMD patients.

molecular biology↗
bioRxiv2022

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

Sickle cell disease (SCD) is due to a mutation in the {beta}-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, a2{beta}S2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling {beta}-globin ({beta}AS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing {beta}AS3-globin and an artificial microRNA (amiR) specifically downregulating {beta}S-globin expression with the aim of reducing HbS levels and favoring {beta}AS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of {beta}S-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells and effective correction of the sickling phenotype, outperforming {beta}AS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile and neither the HSPC viability, engraftment and multi-lineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

genetics↗