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

Abstract

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.

Conflict of interest statement

S.F., B.Y., C.-R.S., N.M., S.L., C.B.R., B.K., A.L., S.B. and W.Y.W. are full-time employees of BioMarin Pharmaceutical and hold stock in BioMarin Pharmaceutical. A.N.M. receives consulting fees from Ambys Medicines, BioMarin Pharmaceutical, HEPATX and Pliant. S.R. reports being an advisory board member for Pfizer, Sanofi, Sigilon and Takeda; receiving conference support from Reliance Life Sciences and Shire/Takeda; and receiving consulting fees from Reliance Life Sciences. G.F.P. reports receiving consulting fees from Ambys Medicines, BioMarin Pharmaceutical, Decibel Therapeutics, Frontera, Intellia, Pfizer, Regeneron, Spark and Third Rock Ventures, and is employed by Voyager Therapeutics. K.J.P. reports receiving consulting fees from Alnylam, Apcintex, BioMarin Pharmaceutical, Bioverativ, Catalyst Bio, Catapult, Chugai, Novo Nordisk, Roche, Sanofi and Sobi; participating as an investigator for BioMarin Pharmaceutical, Sanofi and uniQure; receiving speaker fees from Bayer, BioMarin Pharmaceutical, Biotest, Novo Nordisk, Octapharma, Pfizer, Sanofi, Shire, Sobi and uniQure; and receiving travel support from Alnylam, BioMarin Pharmaceutical, Bayer, Bioverativ, Novo Nordisk, Octapharma, Pfizer, Shire and Sobi. W.L. reports receiving consulting fees from Novo Nordisk, Octapharma and Takeda; participating as an investigator for BioMarin Pharmaceutical; receiving speaker fees from Novo Nordisk, CSL Behring, Sobi and Takeda; and receiving travel support from CSL Behring, Novo Nordisk and Takeda.

© 2022. The Author(s).

Figures

Fig. 1. Histopathology and hFVIII-SQ DNA transduction efficiency in liver biopsy samples from five participants 2.6–4.1 years after gene transfer with valoctocogene roxaparvovec.

a, Representative liver histopathology sections stained with H&E (participants 1, 3, 4 and 11) or hematoxylin and Van Gieson (participant 15); histologic sections per participant were reviewed by the local pathologist and central pathologist, with consistent results: three biopsy levels (participant 1), two levels (participants 11 and 15), one level (participant 3) and four levels (participant 4). Images were captured in QuPath v0.2.3 using the export snapshot feature; no subsequent processing or image enhancement was performed. Scale bars, 180 µm (participant 1); 150 µm (participants 11, 15, 3 and 4) b, Representative liver biopsy sections from each participant showing hFVIII-SQ DNA (brown foci) by ISH. Images were captured at 1,600 × 1,200 pixels and output at 300 pixels per inch (ppi). Each focus (brown dot) represents at least one vector DNA molecule; it is possible to have multiple copies of vector genome within a single focus. Scale bars, 50 µm. c, Percentage of hepatocyte nuclei stained positive for hFVIII-SQ DNA by ISH. Data are means across 11 (participants 1 and 11) or 27–28 (participants 3, 4 and 15) images per biopsy section, spanning ≥50% of the tissue area (biopsy tissue area was larger for participants 3, 4 and 15). Error bars represent the s.e.m., dots represent quantification of each individual image, and data labels show mean values. d, Circular genomes (full length or H–T ITR fused) detected in liver biopsy samples via drop-phase ddPCR following DNA sample treatment with PS-DNase and KpnI (with PS-DNase, all linear forms of DNA are hydrolyzed, and only circular forms of DNA remain; KpnI treatment separates out vector genome units within the concatemeric forms, enabling quantification of genome units within concatemeric vector genomes). e, Qualitative Southern blot analysis of circular episomes after PS-DNase treatment of DNA from liver biopsy samples. Biopsy samples from control, participant 1 and participant 11, and for participants 3, 4 and 15 were processed at separate times; results are presented on separate blots from two independent experiments, and are not intended to present a quantitative comparison. kbp, kilobase pairs; SC, supercoiled markers; W, week. Source data

Fig. 2. Detection of hFVIII-SQ transcript in adult liver biopsy samples.

a, Levels of hFVIII-SQ RNA detected in adult liver samples normalized to endogenous reference RNA (YWHAZ). b, Ratio of hFVIII-SQ RNA to circular full-length DNA (R1–R11-linked DNA, PS-DNase + KpnI). c, Representative images of liver biopsy sections from participants who received a dose of 4 × 103 vg per kg body weight (participants 11 and 15; evaluation of one section each), showing hepatocytes that stained positive for hFVIII-SQ RNA by RISH (arrows indicate cytoplasmic staining of AAV5-FVIII-SQ-derived RNA), and the percentage of hepatocytes staining positive for hFVIII-SQ RNA. Images were captured at 1,600 × 1,200 pixels and output at 300 ppi. Scale bars, 20 µm. d, hFVIII-SQ RNA staining signal per cell in 4 × 103 vg per kg body weight-dosed participants; P = 4.24 × 10−29 for participant 11 (n = 2,212 cells examined in one slide) versus participant 15 (n = 1,397 cells examined in one slide), two-tailed unpaired t-test. YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta. Source data

Fig. 3. Differential gene expression of selected genes potentially mediating low hFVIII-SQ RNA levels in participant 15.

a, Relationship between PHF5A expression and normalized hFVIII-SQ RNA levels in liver biopsy samples of participants (P) who received 4 × 1013 vg per kg body weight (blue) or 6 × 1013 vg per kg body weight (green) valoctocogene roxaparvovec. b, HDAC9 gene expression in participants who received 4 × 1013 vg per kg body weight (blue) or 6 × 1013 vg per kg body weight (green) valoctocogene roxaparvovec. TPM, transcripts per million. Source data

Fig. 4. Expression of GRP78, a regulator of unfolded protein response, in human liver.

a, Representative confocal image of LAMP2, GRP78 and hFVIII protein co-staining from one hepatocyte showing FVIII staining both in the lysosome compartment (indicating FVIII being taken up via the endocytic pathway) and in the ER compartment (FVIII protein expressed from AAV5-hFVIII-SQ). The image is from participant 4. Confocal images were acquired using a Leica TCS Sp8 white-light laser and HyD detectors at 1,024 × 1,024 pixels with an output at 300 ppi. Images were cropped and scale bars added. No additional editing was performed. Representative images of LAMP2, GRP78 and hFVIII protein co-staining from all participant biopsy samples and one normal liver are shown in Extended Data Fig. 7. b, Quantitative analysis of GRP78 intensity per cell (expressing hFVIII-SQ or not in the ER) and correlation between GRP78 intensity per cell and FVIII intensity per cell (Pearson correlation coefficient, r = −0.18; P = 0.04, two-tailed; total of 134 cells from participants 3 (28 cells), 4 (40 cells), 15 (16 cells) and 11 (50 cells), examined over one slide). c, Correlation between GRP78 RNA levels and plasma FVIII activity in the three participants with detectable plasma FVIII activity (Pearson correlation coefficient, r = −0.99; P = 0.018, two-tailed). Participants 1 and 15 had extremely low levels of FVIII RNA and thus very low/undetectable plasma FVIII activity and are therefore not included in this plot. d, GRP78 protein staining by IHC in participants 3, 4 and 11 (representative images from one section per participant). Images were captured at 2,048 × 2,048 pixels and output at 300 ppi. Scale bars, 100 µm. e, Correlation between liver GRP78 RNA and liver GRP78 IHC signals (Pearson correlation coefficient, r = 0.923; P = 0.252, two-tailed; n = 25,887 cells over one slide (participant 3), n = 37,663 cells over one slide (participant 4) and n = 9,091 cells over one slide (participant 11)). f, IHC of endogenous GRP78 protein in 32 normal human liver FFPE samples (one section per individual). Images were captured at 2,048 × 2,048 pixels and output at 300 ppi. Scale bars, 50 µm. Source data

Extended Data Fig. 1. Histopathological assessment of liver biopsy samples from five participants 2.6–4.1 years after gene transfer with valoctocogene roxaparvovec.

Steatosis scored as percentage of lipid-containing hepatocytes.

Extended Data Fig. 2. Valoctocogene roxaparvovec transduction patterns in hepatocytes amongst the four participants with > 2% hepatocytes stained positive.

In situ hybridization analysis to detect vector genomes, showing unbiased distribution of hFVIII-SQ DNA (brown foci) in liver biopsy samples of Participants 11, 15, 3, and 4; Participant 1 has less than 2% of hepatocytes stained positive for vector DNA and did not have any identifiable veins (PC or PV) as the biopsy was broken into many pieces, thus not shown. Each focus (brown dot) represents at least one vector DNA molecule; it is possible to have multiple copies of vector genome within a single focus. Zoom-out (widefield) images allow comparison of liver sections proximal and distal to central veins. Zoom-in images separated according to liver zones – zone 1 being peri-portal vein (PP), zone 3 being peri-central vein (PC), and zone 2 being in between zone 1 and 3. Images were captured at 1600 × 1200 pixels and output at 300 ppi. PV, portal vein.

Extended Data Fig. 3. Quantification of circular vector genomes [DNA] and assessment of multimeric circular episomal forms in liver samples from patients.

Quantification of circular vector genomes [DNA] as measured by ddPCR utilizing primers and probes to detect specific regions of the vector genome: a) R2-R10 linked amplicons. b) R1-R11 linked amplicons (full-length). c) SQ amplicon (representing overall vector genome count). d) ITR-fused amplicons; ITR fusion assay measured 5′ to 3′ ITR recombination (that is head-to-tail, H-T). e) Circular multimeric episome quantification following PS-DNase (eliminates all linear forms of DNA) or PS-DNase/KpnI digestion (to separate individual vector genome units within concatemeric circular forms). ITR fusion assay measured 5′ to 3′ ITR recombination (that is head-to-tail, H-T). f) Qualitative Southern blot assessment of vector genome configuration in circular forms obtained after DNA treatment with PS-DNase and KpnI. DNA fragments corresponding to 3.5 kb, or 4.4 and 2.6 kb could be detected for H-T or H-H/T-T concatemer configurations, respectively. Biopsies from Control, Participant 1, and Participant 11, and for Participants 3, 4, and 15 were processed at separate times; results are presented on separate blots from two independent experiments, and are not intended to present a quantitative comparison. ddPCR, droplet digital polymerase chain reaction; H, head (5′ end); H-H, head-to-head orientation; H-T, head-to-tail orientation; ITR, inverted terminal repeat; kb, kilobase pairs; LM, linear markers; PS-DNase, Plasmid Safe™ ATP-Dependent DNAase; T, tail (3′ end); vg, vector genome. ddPCR was performed after DNA digestion with PS-DNase and KpnI (to eliminate linear forms and to separate individual vector genome units within concatemeric circular forms). Source data

Extended Data Fig. 4. Detection of hFVIII-SQ RNA transcript in patient liver biopsy samples.

hFVIII-SQ RNA levels detected in patient liver samples normalized to endogenous reference RNAs a) beta-actin, b) Rplp0, and c) YWHAZ. d) Correlation of RNA quantified by ddPCR with total RISH area signal in one tissue section per participant; RISH foci sum normalized to tissue area, μm (Pearson correlation coefficient, r = 0.90; p = 0.03, two-tailed). e) Correlation between plasma FVIII activity and liver hFVIII-SQ RNA; Participants 1 and 15 had no or barely detectable plasma FVIII activity (black diamonds). CS, chromogenic substrate assay; hFVIII-SQ, B domain-deleted human factor VIII; P, participant; RISH, RNA in situ hybridization; Rplp0, ribosomal protein lateral stalk subunit P0; YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta. Source data

Extended Data Fig. 5. Gene expression of molecules involved in the regulation of hFVIII-SQ RNA expression.

a) Down-regulation in Participant 15 (low-responder) of molecules involved in positive regulation of transcriptional pathways. b) Upregulation in Participant 15 (low-responder) of zinc and copper metabolic pathway negatively correlated with liver hFVIII-SQ RNA levels. c) Expression levels of transcription factors predicted to bind to the hybrid liver-specific promoter (HLP) in AAV5-hFVIII-SQ. TPM, transcripts per million. Gene names and descriptions: a. Genes clustered in the regulation of transcription, DNA templated pathway. CKS1B, CDC28 protein kinase regulatory subunit 1B; DAB2, Dab, mitogen-responsive phosphoprotein, homolog 2 (Drosophila); DDIT3, DNA-damage-inducible transcript 3; ELK3, ELK3, ETS-domain protein (SRF accessory protein 2); EHF, ets homologous factor; KLF6, Kruppel-like factor 6; PHF5A, plant homeodomain (PHD) finger protein 5A; SMAD3, SMAD family member 3; SS18L1, synovial sarcoma translocation gene on chromosome 18-like 1; TAF8, TAF8 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 43 kDa; APBB1, amyloid beta (A4) precursor protein-binding, family B, member 1 (Fe65); BMP10, bone morphogenetic protein 10; CALCOCO1, calcium binding and coiled-coil domain 1; CAPN3, calpain 3, (p94); CEP290, centrosomal protein 290 kDa; CDK2, cyclin-dependent kinase 2; CRLF3, cytokine receptor-like factor 3; ING5, inhibitor of growth family, member 5; IKBKB, inhibitor of kappa light polypeptide gene enhancer in B cells, kinase beta; IRF1, interferon regulatory factor 1; IRF6, interferon regulatory factor 6; MED17, mediator complex subunit 17; MAPRE3, microtubule-associated protein, RP/EB family, member 3; NUP85, nucleoporin 85 kDa; PLCB1, phospholipase C, beta 1 (phosphoinositide-specific); RNF187, ring finger protein 187; TGFB3, transforming growth factor, beta 3; TP53BP1, tumor protein p53 binding protein 1; ZBTB16, zinc finger and BTB domain containing 16. b. Genes clustered in the Zinc and Copper Metabolism pathway. MT1, metallothionein 1. c. Transcription factors predicted to bind to the HLP promoter in AAV5-hFVIII-SQ. CEBPa, CCAAT/enhancer-binding protein alpha; HNF1a, hepatocyte nuclear factor 1 alpha; HNF4a, hepatocyte nuclear factor 4 alpha. Source data

Extended Data Fig. 6. Log2 normalized expression of all HDAC genes based on responding vs non-responding status.

HDAC RNA expression in non-responder Participant 15 (blue), responders (average of Participants 11, 3, and 4; green), and in naïve human liver (grey). HDAC, histone deacetylase. Source data

Extended Data Fig. 7. Detection of hFVIII protein in human liver and co-localization with organelle-specific markers for lysosomes (LAMP2) and endoplasmic reticulum (GRP78).

LAMP2, GRP78 and hFVIII protein co-staining of hepatocytes from study participant biopsies and one normal liver. Images shown are representative of one section per study participant; the entire biopsy section was reviewed to evaluate hFVIII, LAMP2 and GRP78 co-localization. Images were captured at 2048 × 2048 pixels and output at 300 ppi. LAMP2, lysosome-associated membrane glycoprotein 2; GRP78, glucose-regulated protein 78.