Preclinical evaluation of FLT190, a liver-directed AAV gene therapy for Fabry disease

Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease (Gla-deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of hGLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease

Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors

The absence of an adequate reversal strategy to prevent and stop potential life-threatening bleeding complications is a major drawback to the clinical use of the direct oral inhibitors of blood coagulation factor Xa. Here we show that specific modifications of the substrate-binding aromatic S4 subpocket within the factor Xa active site disrupt high-affinity engagement of the direct factor Xa inhibitors. These modifications either entail amino-acid substitution of S4 subsite residues Tyr99 and/or Phe174 (chymotrypsinogen numbering), or extension of the 99-loop that borders the S4 subsite. The latter modifications led to the engineering of a factor Xa variant that is able to support coagulation in human plasma spiked with (supra-)physiological concentrations of direct factor Xa inhibitors. As such, this factor Xa variant has the potential to be employed to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients

Changes in Dietary Fat Content Rapidly Alters the Mouse Plasma Coagulation Profile without Affecting Relative Transcript Levels of Coagulation Factors

<div><p>Background</p><p>Obesity is associated with a hypercoagulable state and increased risk for thrombotic cardiovascular events.</p><p>Objective</p><p>Establish the onset and reversibility of the hypercoagulable state during the development and regression of nutritionally-induced obesity in mice, and its relation to transcriptional changes and clearance rates of coagulation factors as well as its relation to changes in metabolic and inflammatory parameters.</p><p>Methods</p><p>Male C57BL/6J mice were fed a low fat (10% kcal as fat; LFD) or high fat diet (45% kcal as fat; HFD) for 2, 4, 8 or 16 weeks. To study the effects of weight loss, mice were fed the HFD for 16 weeks and switched to the LFD for 1, 2 or 4 weeks. For each time point analyses of plasma and hepatic mRNA levels of coagulation factors were performed after overnight fasting, as well as measurements of circulating metabolic and inflammatory parameters. Furthermore, in vivo clearance rates of human factor (F) VII, FVIII and FIX proteins were determined after 2 weeks of HFD-feeding.</p><p>Results</p><p>HFD feeding gradually increased the body and liver weight, which was accompanied by a significant increase in plasma glucose levels from 8 weeks onwards, while insulin levels were affected after 16 weeks. Besides a transient rise in cytokine levels at 2 weeks after starting the HFD, no significant effect on inflammation markers was present. Increased plasma levels of fibrinogen, FII, FVII, FVIII, FIX, FXI and FXII were observed in mice on a HFD for 2 weeks, which in general persisted throughout the 16 weeks of HFD-feeding. Interestingly, with the exception of FXI the effects on plasma coagulation levels were not paralleled by changes in relative transcript levels in the liver, nor by decreased clearance rates. Switching from HFD to LFD reversed the HFD-induced procoagulant shift in plasma, again not coinciding with transcriptional modulation.</p><p>Conclusions</p><p>Changes in dietary fat content rapidly alter the mouse plasma coagulation profile, thereby preceding plasma metabolic changes, which cannot be explained by changes in relative expression of coagulation factors or decreased clearance rates.</p></div

Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.

<p>Data are expressed as mean (minimum-maximum expression level).</p><p>*p<0.05 and</p><p>^‡p<0.001 as compared to LFD mice.</p><p>Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.</p

Metabolic parameters of mice on a low fat diet (LFD) or high fat diet (HFD) for 16 weeks as compared to genetically obese <i>ob/ob</i> mice with their littermate wild-type controls after 4 weeks of LFD feeding.

<p>Data are expressed as mean±SEM.</p><p>*p<0.05 and</p><p>^‡p<0.001 as compared to LFD-fed mice or wild-type controls as appropriate.</p><p>Metabolic parameters of mice on a low fat diet (LFD) or high fat diet (HFD) for 16 weeks as compared to genetically obese <i>ob/ob</i> mice with their littermate wild-type controls after 4 weeks of LFD feeding.</p

Effects low fat diet (LFD) and high fat diet (HFD) on plasma coagulation parameters.

<p>Effects on plasma coagulation parameters after 2 (panel A) and 16 (panel B) weeks of low fat diet (white) or high fat diet (black) feeding. Panel C shows the plasma coagulation profile of genetically obese <i>ob/ob</i> mice (striped) and their wild-type littermates (white) after 4 weeks on a low fat diet. Data are presented as mean±SEM. *p<0.05 and ^‡p<0.001 as compared to the LFD-fed mice or wild-type controls as appropriate.</p

Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.

<p>Data are expressed as mean (minimum-maximum expression level).</p><p>*p<0.05 and</p><p>^‡p<0.001 as compared to LFD mice.</p><p>Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.</p