Long-term correction of ornithine transcarbamylase deficiency by WPRE-mediated overexpression using a helper-dependent adenovirus

The urea cycle disorders (UCDs) are important models for developing gene replacement therapy for liver diseases. Long-term correction of the most common UCD, ornithine transcarbamylase (OTC) deficiency, has yet to be achieved in clinical or preclinical settings. The single human clinical trial using early-generation adenovirus (Ad) failed to show any biochemical correction. In adult OTC-deficient mice, an E1/E2-deleted Ad vector expressing the mouse OTC gene, but not the human, was only transiently therapeutic. By using post-transcriptional overexpression in the context of the less immunogenic helper-dependent adenoviral vector, we achieved metabolic correction of adult OTC-deficient mice for \u3e6 months. Demonstrating this result were normalized orotic aciduria, normal hepatic enzyme activity, and elevated OTC RNA and protein levels in the absence of chronic hepatotoxicity. Overexpressing the human protein may have overcome two potential mechanisms accounting for poor cross-species complementation: a kinetic block at the level of mitochondrial import or a dominant negative effect by the mutant polypeptide. These data represent an important approach for treating human inborn errors of hepatocyte metabolism like the UCDs that require high-level transduction and gene expression for clinical correction

198. Tissue restricted overexpression of human ornithine transcarbamylase with a helper-dependent adenovirus containing the WPRE confers long-term correction in OTC-deficient mice

Urea cycle disorders have been models for development of human gene replacement therapy for cell autonomous disorders of hepatocyte metabolism. However, in preclinical studies in mouse models for ornithine transcarbamylase (OTC) deficiency, early generation adenoviral vectors expressing the mouse OTC gene, but not the human, achieved only transient correction (less than 8 weeks). This observation was later attributed to mitochondrial leader peptide sequence differences between the two species which decrease import of human OTC (hOTC), and to chronic toxicity associated with the early generation adenovirus. Moreover, the single human clinical trial failed to show significant clinical correction. We hypothesized that higher hOTC expression, from the less toxic helper-dependent adenoviral vector (HDV), would achieve long term correction in mice and serve as a preclinical model for the efficacy of such a vector in humans. To overexpress hOTC, we incorporated the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and the tissue-restricted phosphoenolpyruvate carboxykinase (PEPCK) promoter into the HDV hOTC vector. Intravenous administration of this vector restored liver OTC activity (as shown by biochemical and histochemical assays) in affected OTCspf-ash mice, and achieved long-term correction of orotic aciduria for at least 6 months (with measurements ongoing). An identical dose of HDV expressing hOTC, but without WPRE, was not efficacious. Southern analysis showed that the therapeutic efficacy of HDV PEPCK hOTC WPRE was not due to increased hepatocyte transduction by vector. Instead, we detected a 9 fold greater steady-state mRNA level, consistent with a posttranscriptional mechanism of enhanced gene expression. Protein levels were also elevated. Hence, by protein amplification, the WPRE was able to effectively increase the level of hOTC expression and overcome the kinetic block at the level of mitochondrial protein import. In conclusion, further development of HDVs using the WPRE for in vivo gene replacement for cell autonomous diseases such as urea cycle disorders is warranted. This vector-transgene combination exhibits decreased long-term toxicity, prolonged and greatly elevated transgene expression, and efficient hepatocyte transduction

In vivo antiviral efficacy of LCTG-002, a pooled, purified human milk secretory IgA product, against SARS-CoV-2 in a murine model of COVID-19

ABSTRACTImmunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosae, with secretory form (sIgA) being dominant and uniquely stable. sIgA is challenging to produce recombinantly but is naturally found in human milk, which could be considered a global resource for this biologic, justifying its development as a mucosal therapeutic. Presently, SARS-CoV-2 was utilized as a model mucosal pathogen, and methods were developed to efficiently extract human milk sIgA from donors who were naïve to SARS-CoV-2 or had recovered from infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA in their milk (pooled to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1% or greater were all associated with sIgA. Western blot demonstrated that all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher Spike binding (mean endpoint of 0.87 versus 5.87). LCTG-002 was capable of blocking the Spike receptor-binding domain – angiotensin-converting enzyme 2 (ACE2) interaction with significantly greater potency compared to control (mean LCTG-002 IC50 154ug/mL versus 50% inhibition not achieved for control), and exhibited significant neutralization activity against Spike-pseudotyped virus infection (mean LCTG-002 IC50 49.8ug/mL versus 114.5ug/mL for control). LCTG-002 was tested for its capacity to reduce viral lung burden in K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 significantly reduced SARS-CoV-2 titers compared to control when administered at 0.25 mg/day or 1 mg/day, with a maximum TCID50 reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure and efficacious in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics

Effect of Thiol Pendant Conjugates on Plasmid DNA Binding, Release, and Stability of Polymeric Delivery Vectors

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell’s nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly­(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer–plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex–protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release