Induction of ER Stress by an AAV5 BDD FVIII Construct Is Dependent on the Strength of the Hepatic-Specific Promoter

Fong, Sylvia; Handyside, Britta; Sihn, Choong-Ryoul; Liu, Su; Zhang, Lening; Xie, Lin; Murphy, Ryan; Galicia, Nicole; Yates, Bridget; Minto, Wesley; Vitelli, Catherine; Harmon, Danielle; Ru, Yuanbin; Yu, Guoying Karen; Escher, Claudia; Vowinckel, Jakob; Woloszynek, Jill; Akeefe, Hassib; Mahimkar, Rajeev; Bullens, Sherry; Bunting, Stuart

doi:10.1016/j.omtm.2020.07.005

Cited by 27 publications

(23 citation statements)

References 45 publications

(85 reference statements)

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“… 30 , 31 Studies in mice have shown that hepatocytes expressing B-domain-deleted FVIII (from vectors incorporating a stronger hepatic-specific promoter than is used in valoctocogene roxaparvovec) may mount an unfolded protein response, potentially inducing ER stress. 32 – 34 To investigate the possibility of ER stress in our samples, fluorescence signals for GRP78 and FVIII protein colocalized in ER were quantified on a per-cell basis. Within individual samples, hepatocytes with detectable hFVIII-SQ protein staining in the ER did not have elevated expression of GRP78 compared to hepatocytes without hFVIII-SQ staining in the ER (Fig.…”

Section: Resultsmentioning

confidence: 99%

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Fong

Yates

Sihn

et al. 2022

Nat Med

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Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial (NCT02576795), liver biopsy samples were collected 2.6–4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability.

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Section: Resultsmentioning

confidence: 99%

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Fong

Yates

Sihn

et al. 2022

Nat Med

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“…We propose that simply increasing FVIII expression in transduced cells may not be sufficient to overcome the poor durability of HA gene therapy and may even induce potential unwanted off-target and toxic responses. This view is further supported by a recent publication, showing that the overexpression BDD-FVIII in murine hepatocytes from an AAV vector induces the UPR (61), which may be avoided by achieving therapy through limited expression in a high percentage of hepatocytes.…”

Section: Discussionmentioning

confidence: 79%

“…Moreover, we demonstrate a role of diet in particular when expressing a therapeutic protein in the liver that is prone to misfolding and ER stress. While AAV-FVIII gene transfer has not been found to cause the level of hepatocyte apoptosis and liver injury that may occur with DNA vectors, multiple studies have shown induction of ER stress markers in mice in response to AAV-FVIII (50, 59, 60), and liver injury (albeit of unknown origin) occurred in patients treated with high-dose AAV-FVIII (9, 10) We have now directly shown that expression of SQ-BDD in primary human hepatocytes induced ER stress in a dose-dependent manner (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

Ectopic FVIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma

Deng

Kapelanski-Lamoureux

Gao

et al. 2021

Preprint

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Clotting Factor VIII (FVIII) is the protein deficient in hemophilia A (HA), an X chromosome-linked bleeding disorder affecting 24.6 per 100,000 males at birth. Scientists and clinical physicians have been striving hard to find a cure for this disease. Presently, HA therapy involves prophylactic infusion of plasma-derived or recombinant-derived FVIII. Recent clinical studies have reported success using recent adeno-associated-virus (AAV) vector mediated delivery of a FVIII gene. FVIII is a 330 kDa glycoprotein comprised of three domains (A1-A2-B-A3-C1-C2). The B domain is dispensable and B domain-deleted (BDD) FVIII is used in the clinic as an effective protein replacement therapy for HA. Previously, we observed that hydrodynamic tail vein injection of the BDD-FVIII vector resulted in FVIII aggregation and endoplasmic reticulum (ER) stress in murine livers. Additionally, mice that were exposed to transient ER stress and then fed a high fat diet (HFD) for 9 months developed hepatocellular carcinoma (HCC). Therefore, we deduced that ectopic transient expression of FVIII in hepatocytes of mice with subsequent HFD feeding may also develop HCC. Here, we tested hepatocyte expression of two BDD-FVIII variants in vivo. BDD-FVIII aggregates in the ER and induces hepatocyte apoptosis, and N6-FVIII is more efficiently folded and secreted from the ER and causes less hepatocyte apoptosis. We performed tail vein injections of vectors directing expression of BDD-FVIII or N6-BDD to hepatocytes of 6-week old mice. One week after injection, mice were fed 60% HFD for 65 weeks. We found that 50% of mice that received N6-BDD developed liver tumors; in contrast, 90% of mice given BDD-FVIII developed liver tumors. Of the mice injected with BDD-FVIII, 30% developed carcinomas and 60% developed adenomas. Similar masses were not observed in mice that received empty vector. These findings raise concerns about the safety of long-term ectopic expression of FVIII in hepatocytes during FVIII gene therapy

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“…Moreover, the production of AAV vectors with genomes >4.7 kb is challenging because they frequently contain truncated genomes during viral packaging and are unsuitable for full-length FVIII cDNA (10.6 kb) or even B-domain deleted FVIII cDNA (4.35 kb) gene delivery [ 48 ]. A previous report also showed that some patients may generate neutralizing antibodies or endoplasmic reticulum stress, which influences the treatment effect of AAV-based gene delivery [ 27 ]. Therefore, lipid-coated nanoparticles, such as PEI and DPPC, have been developed as nonviral delivery systems to transport DNA or drugs into cells.…”

Section: Discussionmentioning

confidence: 99%

“…A first-in-human clinical trial of an AAV5- hFVIII -SQ vector for treating 15 adults with severe hemophilia A concluded that the therapy was beneficial and safe [ 26 ]. However, the foreign gene size is limited in the AAV vector, and some patients will generate neutralizing antibodies or endoplasmic reticulum stress to influence the treatment effect [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles

et al. 2021

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Hemophilia A is a bleeding disease caused by loss of coagulation factor VIII (FVIII) function. Although prophylactic FVIII infusion prevents abnormal bleeding, disability and joint damage in hemophilia patients are common. The cost of treatment is among the highest for a single disease, and the adverse effects of repeated infusion are still an issue that has not been addressed. In this study, we established a nonviral gene therapy strategy to treat FVIII knockout (FVIII KO) mice. A novel gene therapy approach was developed using dipalmitoylphosphatidylcholine formulated with iron oxide (DPPC-Fe3O4) to carry the B-domain-deleted (BDD)-FVIII plasmid, which was delivered into the FVIII KO mice via tail vein injection. Here, a liver-specific albumin promoter-driven BDD-FVIII plasmid was constructed, and the binding ability of circular DNA was confirmed to be more stable than that of linear DNA when combined with DPPC-Fe3O4 nanoparticles. The FVIII KO mice that received the DPPC-Fe3O4 plasmid complex were assessed by staining the ferric ion of DPPC-Fe3O4 nanoparticles with Prussian blue in liver tissue. The bleeding of the FVIII KO mice was improved in a few weeks, as shown by assessing the activated partial thromboplastin time (aPTT). Furthermore, no liver toxicity, thromboses, deaths, or persistent changes after nonviral gene therapy were found, as shown by serum liver indices and histopathology. The results suggest that this novel gene therapy can successfully improve hemostasis disorder in FVIII KO mice and might be a promising approach to treating hemophilia A patients in clinical settings.

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Induction of ER Stress by an AAV5 BDD FVIII Construct Is Dependent on the Strength of the Hepatic-Specific Promoter

Cited by 27 publications

References 45 publications

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Ectopic FVIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma

Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles

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