Human gene therapy最新文献

Seven cases of hematological malignancy reported in recipients of Skysona™ (elivaldogene autotemcel) have reignited long-standing concerns about insertional mutagenesis in lentiviral vector (LV)-based gene therapy. Here, we dissect the molecular and clinical evidence underlying these events, place them in the broader context of over 300 patients treated with LV-modified hematopoietic stem and progenitor cells (HSPCs), and review the real-world safety record of LV-engineered chimeric antigen receptor T cells. We show that cancers associated with Skysona are mechanistically linked to the use of a potent viral MNDU3 promoter probably combined with intensive conditioning and growth-factor support, whereas LV products employing weak or physiological promoters continue to display an excellent safety profile. With event rates <0.6/100 patient-years, lower than those after autologous HSCT, the therapeutic index of approved LV-HSPC advanced therapy medicinal products remains favorable. Ongoing optimization of vector design, conditioning, and long-term surveillance, together with emerging genome-editing platforms, is expected to further mitigate residual risk.

The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. While CRISPR-based CFTR editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and organoid cultures derived from patients with CF, their efficacy remains suboptimal. Here, we developed the CuFi^{Cas9(Y66S)eGFP} reporter system by integrating spCas9 and a non-fluorescent Y66S eGFP mutant into CuFi-8 cells, an immortalized human airway epithelial cell line derived from a patient with CF with homozygous F508del mutations. These cells retain the basal cell phenotype in proliferating cultures and can differentiate into polarized airway epithelium at an air-liquid interface (ALI), enabling both visualized detection of gene editing and electrophysiological assessment of CFTR functional restoration. Using this system, recombinant adeno-associated virus (rAAV)-mediated homology-directed repair (HDR) was evaluated in proliferating cultures. A correction rate of 13.5 ± 0.8% was achieved in a population where 82.3 ± 5.6% of cells were productively transduced by AAV.eGFP630g2-CMVmCh, an rAAV editing vector with an mCherry reporter. Dual-editing of F508del CFTR and Y66S eGFP was explored using AAV.HR-eGFP630-F508(g03) to deliver two templates and single guide RNAs. eGFP⁺ (Y66S-corrected) cells and eGFP^- (non-corrected) cells were sorted via fluorescence-activated cell sorting and differentiated at an ALI to assess the recovery of CFTR function. Despite a low F508 correction rate of 2.8%, ALI cultures derived from the eGFP^- population exhibited 25.2% of the CFTR-specific transepithelial Cl^- transport observed in CuFi-ALI cultures treated with CFTR modulators. Next-generation sequencing revealed frequent co-editing at both genomic loci, with sixfold higher F508 correction rate in the eGFP⁺ cells than eGFP^- cells. In both populations, non-homology end joining predominated over HDR. This reporter system provides a valuable platform for optimizing editing efficiencies in proliferating airway basal cells, particularly for development of strategies to enhance HDR through modulation of DNA repair pathways.

Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntington's disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering and whether these changes can be measured in peripheral biofluids. We previously found that lipid changes that occur in brain with HD progression could be prevented by attenuating HTT transcription of the mutant allele in a genetic mouse model (LacQ140) with inducible whole body lowering. Here, we tested whether intrastriatal injection of a therapeutic capable of repressing the mutant HTT allele with expanded cytosine-adenine-guanine (CAG) can provide similar protection against lipid changes in HD mice with a deletion of neo cassette (zQ175DN). Wild-type or zQ175DN mice were injected with adeno-associated virus 9 (AAV9) bearing a cDNA for a zinc finger protein (ZFP), which preferentially targets mutant HTT (ZFP-HTT) to repress transcription. Proteins from brain tissues were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses of brain tissue and plasma collected from the same mice were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic instability index was assessed using capillary gel electrophoresis of PCR products and was shown to be impeded by ZFP-HTT. Lowering mHTT levels by 43% for 4 months prevented loss of total lipid content including the subclasses sphingomyelin, ceramide, phosphatidylethanolamine and others of caudate-putamen in zQ175DN mice. Moreover, LC-MS analysis of plasma demonstrated total lipid increases and lipid changes in monogalactosyl monoacylglyceride and certain phosphatidylcholine species were reversed with the therapy. In summary, our data demonstrate that analyzing lipid signatures of brain tissue and peripheral biofluids are valuable approaches for evaluating potential therapies in a preclinical model of HD.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). While gene therapy holds promise as a cure, the cell-type-specific heterogeneity of CFTR expression in the lung presents significant challenges. Current CF ferret models closely replicate the human disease phenotype but have limitations in studying functional complementation through cell-type-specific CFTR restoration. To address this, we developed a new transgenic ferret line, CFTR^{int1-eGFP(lsl)}, in which a Cre-recombinase (Cre)-excisable enhanced fluorescent protein (eGFP) reporter cassette is knocked in (KI) to intron 1 of the CFTR locus. Breeding this reporter line with CFTR^G551D CF ferret resulted in a novel CF model, CFTR^{int1-eGFP(lsl)/G551D}, with disease onset manageable via the administration of CFTR modulator VX770. In this study, we confirmed two key properties of the CFTR^{int1-eGFP(lsl)/G551D} CF ferrets: (1) cell-type-specific expression of the CFTR(N-24)-eGFP fusion protein, driven by the intrinsic CFTR promoter, in polarized epithelial cultures and selected tissues, and (2) functional reversion of the KI allele via Cre-mediated excision of the reporter cassette. This model provides a valuable tool for studying the effects of targeted CFTR reactivation in a cell-type-specific manner, which is crucial for enhancing our understanding of CFTR's roles in modulating airway clearance and innate immunity, and for identifying relevant cellular targets for CF gene therapy.

Chronic kidney disease (CKD) is a major global health problem characterized by renal fibrosis, for which effective therapeutic options are still lacking. Mesenchymal stem cells (MSCs) have emerged as potential candidates for treating fibrosis due to their paracrine effects. This study first compared the antifibrotic capacities of umbilical cord-derived MSCs (UCMSCs) and dental pulp stem cells (DPSCs). The results showed that DPSCs exhibited superior effects in suppressing fibrosis markers and improving the fibrotic microenvironment. Thus, subsequent studies focused on DPSC and their hepatocyte growth factor (HGF)-modified counterpart (HGF-DPSC). Using an in vivo unilateral ureteral obstruction (UUO) mouse model and an in vitro Transforming Growth Factor-Beta 1(TGF-β1)-induced Human Renal Proximal Tubule Epithelial Cell (HK-2 cell) model, this study systematically evaluated the promising antifibrotic effects and mechanisms of DPSC. The results demonstrated that HGF-DPSC significantly improved the fibrotic microenvironment by regulating the Phosphoinositide 3-Kinase/Protein Kinase B/Glycogen Synthase Kinase 3 Beta (PI3K/AKT/GSK3β) signaling pathway and suppressing β-catenin activation. We confirmed direct protein-protein interaction between HGF and Iodothyronine Deiodinase 2 (DIO2) through co-immunoprecipitation (Co-IP), which suggested a novel molecular mechanism by which HGF-DPSC exerts its antifibrotic effects. These findings highlight the multitarget mechanism of HGF-DPSC in the treatment of renal fibrosis and provide new insights and possibilities for the treatment of CKD.

Recombinant adeno-associated virus (rAAV) has emerged as a leading vehicle for human gene therapy. An accurate and precise infectious titer assay is critical for assessing rAAV quality, potency, and product stability. The current gold standard for measuring rAAV infectivity is the median tissue culture infectivity dose (TCID50) method, which is laborious and highly variable. In the past several years, the droplet digital PCR (ddPCR) technology has made profound impacts on gene therapy analytics as it provides absolute DNA copy quantitation and is more accurate and precise than qPCR. In this article, we leveraged the ddPCR technology and developed a method to quantify rAAV cellular uptake in vitro. The results demonstrated that our method is consistent with TCID50 but is significantly more precise. Utilizing a stable AAV receptor (AAVR) cell line, this method can be implemented as a platform approach for various AAV serotypes and target genes. Moreover, the method is stability indicating, as desired for a potency assay. In conclusion, a novel rAAV uptake assay has been developed which reflects the mechanism of action of rAAV, and is accurate, precise and sensitive to product quality; thus overcoming many of the challenges of the traditional TCID50 method. It is particularly useful for initial rAAV product quality assessment and can contribute to a robust assay matrix with other product-specific potency assays for late-stage programs.