Molecular Therapy-Methods & Clinical Development最新文献

The COVID-19 pandemic has caused about seven million deaths worldwide. Preventative vaccines have been developed including Spike gp mRNA-based vaccines that provide protection to immunocompetent patients. However, patients with primary immunodeficiencies, patients with cancer, or hematopoietic stem cell transplant recipients are not able to mount robust immune responses against current vaccine approaches. We propose to target structural SARS-CoV-2 antigens (i.e., Spike gp, Membrane, Nucleocapsid, and Envelope) using circulating human antigen presenting cells electroporated with full length SARS-CoV-2 structural protein-encoding mRNAs to activate and expand specific T cells. Based on the Th1-type cytokine and cytolytic enzyme secretion upon antigen rechallenge, we were able to generate SARS-CoV-2 specific T cells in up to 70% of unexposed unvaccinated healthy donors (HDs) after 3 subsequent stimulations and in 100% of recovered patients (RPs) after 2 stimulations. By means of SARS-CoV-2 specific TCRβ repertoire analysis, T cells specific to Spike gp-derived hypomutated regions were identified in HDs and RPs despite viral genomic evolution. Hence, we demonstrated that SARS-CoV-2 mRNA-loaded antigen presenting cells are effective activating and expanding COVID19-specific T cells. This approach represents an alternative to patients who are not able to mount adaptive immune responses to current COVID19 vaccines with potential protection across new variants that have conserved genetic regions.

Friedreich’s ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein, frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of FRDA’s cardiac phenotype was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of the FXN gene. The Fxn^flox/null::MCK-Cre conditional knockout mouse (FXN-MCK) has a FXN gene ablation that prevents frataxin expression in cardiac and skeletal muscle, leading to cardiac insufficiency, weight loss and morbidity. FXN-MCK mice received a single intravenous injection of an AAV8 vector containing human (hFXN) or mouse (mFXN) FXN gene under the control of a phosphoglycerate kinase promoter. Compared to vehicle-treated FXN-MCK control mice, AAV-treated FXN-MCK mice displayed increases in body weight, reversal of cardiac deficits and increases in survival without apparent toxicity in the heart or liver for up to 12 weeks post dose. Frataxin protein expression in heart tissue was detected in a dose-dependent manner, exhibiting wide distribution throughout the heart similar to wild-type, but more speckled. These results support an AAV8-based approach to treat FRDA-associated cardiomyopathy.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms were rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped Vesicular Stomatitis Virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 + 0.42 ×10⁶ cells/mL in 1 L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 + 0.58 ×10⁸ plaque-forming units (pfu)/mL at 3 days post-infection. When compared to growth in plate-based cultures this was a 29-fold increase in virus production, meaning a 1 L bioreactor produces the same amount of virus as 1284 15cm plates. Additionally, the omicron BA.1 S-VSV reached a peak titer of 5.58 + 0.35 ×10⁶ pfu/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-pfu ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Despite the implementation of life-saving newborn screening programs and a galactose-restricted diet, many patients with Classic Galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which not only decreased galactose-1 phosphate (gal-1P) in the liver, but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, non-invasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of Galactosemia. While our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that target the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compounds in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV-depletion from the extracellular medium restored transfection efficiency in conditions suffering from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UGCG enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV-depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12x10⁶ cells/mL. These results suggest that the interaction between polyplexes and extracelluar and intracellular vesicles plays a crutial role in the CDE, providing insights for the development of strategies to mitigate its impact.

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. This work studied the effect of the genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension adapted HEK293 cells via triple transfection using AAV plasmids containing the same green fluorescent protein (GFP) transgene with DNA stuffers for variable size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography and the results suggest that the triple transfection condition significantly impacts the AEX retention time and resolution between the full and empty capsid peaks. Charge detection mass spectrometry (CD-MS) was performed on all AEX full capsid peak samples showing a wide distribution of empty, partial, full length, and co-packaged DNA in the capsids. The AEX purified samples were then analyzed by differential scanning fluorimetry (DSF) and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.