Molecular Therapy-Methods & Clinical Development最新文献

Mucopolysaccharidosis type IVB (MPSIVB) is a lysosomal storage disorder caused by β-galactosidase (β-GAL) deficiency characterized by severe skeletal and neurological alterations without approved treatments. To develop hematopoietic stem progenitor cell-gene therapy (HSPC-GT) for MPSIVB, we designed lentiviral vectors (LVs) encoding human β-GAL to achieve supraphysiological release of the therapeutic enzyme in human HSPCs and metabolic correction of diseased cells. Transduced HSPCs displayed proper colony formation, proliferation, and differentiation capacity, but their progeny failed to release the enzyme at supraphysiological levels. Therefore, we tested alternative LVs to overexpress an enhanced β-GAL deriving from murine (LV-enhGLB1) and human selectively mutated GLB1 sequences (LV-mutGLB1). Only human HSPCs transduced with LV-enhGLB1 overexpressed β-GAL in vitro and in vivo without evidence of overexpression-related toxicity. Their hematopoietic progeny efficiently released β-GAL, allowing the cross-correction of defective cells, including skeletal cells. We found that the low levels of human GLB1 mRNA in human hematopoietic cells and the improved stability of the enhanced β-GAL contribute to the increased efficacy of LV-enhGLB1. Importantly, the enhanced β-GAL enzyme showed physiological lysosomal trafficking in human cells and was not associated with increased immunogenicity in vitro. These results support the use of LV-enhGLB1 for further HSPC-GT development and future clinical translation to treat MPSIVB multisystem disease.

Macrophage-based cell therapeutics are an emerging modality to treat cancer and repair tissue damage. A reproducible manufacturing and engineering process is central to fulfill their therapeutical potential. Here, we establish a robust macrophage manufacturing platform (Mo-Mac), and demonstrate that macrophage functionality can be enhanced by N1-methylpseudouridine (m1Ψ)-modified mRNA. Using single-cell transcriptomic analysis as an unbiased approach, we found that >90% cells in the final product were macrophages, and the rest primarily comprised T cells, B cells, natural killer cells, promyelocytes, promonocytes and hematopoietic stem cells. This analysis also guided the development of flow cytometry strategies to assess cell compositions in the manufactured product to meet requirements by the National Medical Products Administration. To modulate macrophage functionality, as an illustrative example, we examined whether the engulfment capability of macrophages could be enhanced by mRNA technology. We found that efferocytosis was increased in vitro when macrophages were electroporated with m1Ψ-modified mRNA encoding CD300LF (CD300LF-mRNA-macrophage). Consistently, in a mouse model of acute liver failure, CD300LF-mRNA-macrophage facilitated organ recovery from acetaminophen-induced hepatotoxicity. These results demonstrate a GMP-compliant macrophage manufacturing process, and indicate that macrophage can be engineered by versatile mRNA technology to achieve therapeutic goals.

Lamellar Ichthyosis (LI) is a chronic disease, mostly caused by mutations in TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene edited cells showed complete absence of Transglutaminase-1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a SINγ-RV vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity and cornified envelopes formation, hence restored proper epidermal architecture. Single cell multiomic analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial to epidermal regeneration. This study serves as a proof-of-concept for assessing the potential of this therapeutic approach in treating TGM1 dependent LI.

Standardised evaluation of adeno-associated virus (AAV) vector products for biotherapeutic application is essential to ensure the safety and efficacy of gene therapies. This includes analysing the critical quality attributes of the product. However, many of the current analytical techniques used to assess these attributes have limitations, including low-throughput, large sample requirements, poorly understood measurement variability, and lack of comparability between methods. To address these challenges, it is essential to establish higher-order reference methods that can be used for comparability measurements, optimisation of current assays, and development of reference materials. Highly precise methods are necessary for measuring the empty/partial/full capsid ratios and the titre of AAV vectors. Additionally, it is important to develop methods for the measurement of less established critical quality attributes, including post-translational modifications, capsid stoichiometry, and methylation profiles. By doing so, we can gain a better understanding of the influence of these attributes on the quality of the product. Moreover, quantification of impurities, such as host cell proteins and DNA contaminants, is crucial for obtaining regulatory approval. The development and application of refined methodologies will be essential to thoroughly characterise AAV vectors, by informing process development and facilitating the generation of reference materials for assay validation and calibration.

Adenoviruses (Ad) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector “ChAdOx1” in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the Coxsackie and Adenovirus Receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of a RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumour-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.

In a recent Phase IIa clinical trial, the candidate leishmaniasis vaccine ChAd63-KH was shown to be safe and immunogenic in Sudanese patients with post kala- azar dermal leishmaniasis. However, its value as a stand-alone therapeutic was unknown. To assess the therapeutic efficacy of ChAd63-KH, we conducted a randomized, double-blind, placebo-controlled Phase IIb trial (Clinicaltrials.gov registration: NCT03969134). Primary outcomes were safety and efficacy (≥90% improvement in clinical disease). Secondary outcomes were change in severity grade and vaccine-induced immune response. 86 participants with uncomplicated post kala azar dermal leishmaniasis of ≥ six months duration were randomised to receive ChAd63-KH (7.5x10¹⁰ viral particles once i.m.) or placebo. 75 participants (87%) completed the trial as per protocol. No severe or serious adverse events were observed. At day 90 post vaccination, 6/40 (15%) and 4/35 (11%) participants in the vaccine and placebo groups respectively showed ≥ 90% clinical improvement (RR 1.31 [95% CI, 0.40 to 4.28], p=0.742). There were also no significant differences in PKDL severity grade between study arms. Whole blood transcriptomic analysis identified transcriptional modules associated with interferon responses and monocyte and dendritic cell activation. Thus, a single vaccination with ChAd63-KH showed no therapeutic efficacy in this subset of Sudanese PKDL patients.

Targeted gene editing to restore CD40L expression via homology-directed repair (HDR) in CD34⁺ hematopoietic stem and progenitor cells (HSPCs) represents a potential long-term therapy for X-linked hyper IgM syndrome. However, clinical translation of HSPC editing is limited by inefficient long-term engraftment of HDR-edited HSPCs. Here, we ameliorate this issue by employing a small-molecule inhibitor of DNA-PKcs, AZD7648, to bias DNA repair mechanisms to facilitate HDR upon CRISPR SpCas9-based gene editing. Using AZD7648 treatment and a clinically relevant HSPC source, mobilized peripheral blood CD34⁺ cells, we achieve ∼60% HDR efficiency at the CD40LG locus and enhanced engraftment of HDR-edited HSPCs in primary and secondary xenotransplants. Specifically, we observed a 1.6-fold increase of HDR-edited long-term HSPCs in primary transplant recipients without disturbing chimerism levels or differentiation capacity. As CD40L is primarily expressed in T cells, we demonstrate T cell differentiation from HDR-edited HSPCs in vivo and in artificial thymic organoid cultures, and endogenously regulated CD40L expression following activation of in-vivo-derived CD4⁺ T cells. Our combined findings demonstrate HDR editing at the CD40LG locus at potentially clinically beneficial levels. More broadly, these data support using DNA-PKcs inhibition with AZD7648 as a simple and efficacious addition to HSPC editing platforms.

Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, bio-layer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalization of the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet® AAVX biosensors across four rAAV serotypes (rAAV2, 5, 8, and 9) and applied in an upstream and downstream processing context. AAVX-BLI measured capsid titer across a wide concentration range (1×10¹⁰ – 1×10¹² capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS™ CaptureSelect™ AAVX affinity chromatography, showing resin breakthrough dependence on operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.

With more than 130 clinical trials and eight approved gene therapy products, AAVs stand as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as micro-RNAs, has not been previously investigated. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the rAAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 10⁹ and 10¹¹ vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p﹤0.05, t-test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.