Journal: Journal of Inherited Metabolic Disease

Article Title: Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

doi: 10.1007/s10545-017-0053-3

Figure Lengend Snippet: Clinical trials of gene therapy products for liver monogenic disorders. Intraportal and intrahepatic routes of administration relate to injection on the portal vein and the hepatic artery respectively. FVIII: factor VIII; FIX: factor IX; HD-adenovirus: helper-dependent adenovirus; LDL: low density lipoprotein; MoML: Moloney murine leukaemia virus; NA: not applicable; OTC: ornithine transcarbamylase; PBGD: porphobilinogen deaminase. Information sources: clinicaltrial.gov (accessed 06/01/2017) and company websites. If the date of the start of the trial was not available in clinicaltrials.gov website, the date of publication of the results is mentioned. The list of trials announced for 2017 is indicative and does not pretend to be exhaustive

Article Snippet: After a single intravenous injection of AAV vectors with different capsids encoding the FIX gene or its Padua FIX variant, which contains a gain-of-function mutation, reported stabilised plasma FIX levels have ranged from 3 to 8% in the AMT-060 trial sponsored by Uniqure (Miesbach et al ) and the DTX-101 sponsored by Dimension Therapeutics ( http://dimensiontx.com ) to 20–44% in the high-dose cohort of the BAX 335 trial sponsored by Shire (Monahan et al ) and in the SPK-9001 trial sponsored by Spark Therapeutics/Pfizer (George et al ).

Techniques: Injection, Plasmid Preparation, Ex Vivo, Mutagenesis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Meganuclease targeting HSV-1 protects against herpetic keratitis: Application to corneal transplants

doi: 10.1016/j.omtn.2022.11.006

Figure Lengend Snippet: Meganuclease targeting HSV-1 protects against endothelial infection in an in vivo rabbit herpetic keratitis model (A) In vivo endothelium transduction efficiency after intracameral adeno-associated-virus-enhanced green fluorescent protein (AAV-EGFP) injection. Macroscopic examination for reporter direct fluorescence (top) and DAPI staining (bottom) 2 weeks after vector injection (n = 6). Scale bar, 50 μm. (B–E) Analysis of endothelial X-Gal-generated chromophore (X-Gal-c) release 40 h after rHSV-1 infection. Two weeks after intracameral injection of AAV-control (AAV-CTRL)- or meganuclease (AAV-MEGA)-vector suspension, the anterior chamber was inoculated with 3 × 10 4 or 8 × 10 4 rHSV-1 PFU (four experiments in the 3 × 10 4 -PFU group samples n = 13; three experiments in the 8 × 10 4 -PFU group with nine controls and eight test samples). (B) Macroscopic endothelial X-Gal-c signals. X-Gal-c-positive clusters of infected endothelial cells (plaque) in AAV-CTRL- or AAV-MEGA-treated corneas. Scale bars, 1 mm (left) and 250 μm (right). (C) Endothelial plaque-subtype analysis. X-Gal-c release (top) and DAPI staining (bottom). 1: endothelial plaque; 2: keratic precipitate (KP)-associated plaque; 3, 4: eroded plaques. Scale bar, 250 μm. (D) Endothelial plaque and subtype quantification. ImageJ analysis using the outlines of endothelial X-Gal-c release in AAV-CTRL- (white columns) and AAV-MEGA-treated (black columns). Results are expressed as mean ± SD and percentage ± SD for representative experiments (n = 8 for each group). Two-sided Mann-Whitney test or Kolmogorov-Smirnov (D) test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (E) Individual plaque-area analysis (including eroded plaques) after rHSV-1 infection (six control and test corneas of the first experiments). (F) qPCR rHSV-1 genome/quantification in the aqueous humor 40-h postinfection with 8 × 10 4 rHSV-1 PFU (eight controls and seven test eyes). Two-sided Mann-Whitney test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G) Corneal slit-lamp photographs postinfection with 3000 rHSV-1 PFU (top) or 8 ×10 4 PFU (bottom) showing broad and magnified representative views of endothelial KPs and focal corneal edema (scale bar, 2 mm). The presence of KPs and edema was scored 0 or 1 in each of the five corneal zones and their sums are represented schematically by sector (right). Fisher’s exact test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: After selecting a viral vector adequate for corneal endothelium transduction , experiments involved single-stranded AAVs based on the AAV-2 genome, equipped with an expression cassette of an intervening sequence (produced as previously described ): (1) coding for the EGFP [AAV-EGFP]; (2) coding for a meganuclease directed against either the ICP0 gene (HSV-1m4) or the capsid protein VP5 gene encoding UL19 (HSV-1m2; AAV-MEGA) ; (3) noncoding EGFP sequence in antisense orientation, with three stop codons close to the promoter (AAV-CTRL), driven by the human ECMV promoter, and packaged in an AAV-1 capsid (vectors AAV-2/1) (Vectalys).

Techniques: Infection, In Vivo, Transduction, Virus, Injection, Fluorescence, Staining, Plasmid Preparation, Generated, Control, Suspension, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Meganuclease targeting HSV-1 protects against herpetic keratitis: Application to corneal transplants

doi: 10.1016/j.omtn.2022.11.006

Figure Lengend Snippet: Meganuclease-treated corneal grafts are protected against rHSV-1 endothelial infection in an in vivo rabbit cornea-transplant model Donor corneal buttons were cultured with adeno-associated-virus control (AAV-CTRL)- or meganuclease (AAV-MEGA)-coding vector. Recipient corneas were trephined and their central portion removed. Transduced donor corneas were trephined and sutured to the recipient corneal bed. Four weeks later, eyes were injected intracamerally with 9 × 10 4 rHSV-1 PFU. Corneal herpetic infections were analyzed 40 h after postinoculation (n = 5 in each group). (A and top panel B) Macroscopic image of endothelial X-Gal-generated chromophore (X-Gal-c) release. Scale bars, 2 mm (A) and 250 μm (B). Dotted red lines represent graft suture lines containing fibrotic tissue. (B bottom panel) DAPI staining. (C) Endothelial plaque counts, subtype percentage, and total plaque-area analyses based on endothelial X-Gal-c release and qPCR determined rHSV-1 genome numbers. Infection area includes X-Gal-c-colored areas and eroded surfaces. Results are expressed as mean ± SD (n = 5 in each group). Two-sided Mann-Whitney test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) Slit-lamp photographs showing broad and magnified representative views of endothelial keratic precipitates (KPs) and focal corneal edema (scale bar, 2 mm). (E) Schematic diagrams of KP quantification and corneal edema (n = 5 in each group; Fisher’s exact test; ∗p < 0.05.

Article Snippet: After selecting a viral vector adequate for corneal endothelium transduction , experiments involved single-stranded AAVs based on the AAV-2 genome, equipped with an expression cassette of an intervening sequence (produced as previously described ): (1) coding for the EGFP [AAV-EGFP]; (2) coding for a meganuclease directed against either the ICP0 gene (HSV-1m4) or the capsid protein VP5 gene encoding UL19 (HSV-1m2; AAV-MEGA) ; (3) noncoding EGFP sequence in antisense orientation, with three stop codons close to the promoter (AAV-CTRL), driven by the human ECMV promoter, and packaged in an AAV-1 capsid (vectors AAV-2/1) (Vectalys).

Techniques: Infection, In Vivo, Cell Culture, Virus, Control, Plasmid Preparation, Injection, Generated, Staining, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Meganuclease targeting HSV-1 protects against herpetic keratitis: Application to corneal transplants

doi: 10.1016/j.omtn.2022.11.006

Figure Lengend Snippet: Intrastromal injection of the meganuclease-coding vector into in vivo rabbit corneas inhibited rHSV-1 corneal infection (A) Enhanced green fluorescent protein (EGFP) expression following intrastromal injection of adeno-associated virus (AAV)-EGFP. Reporter direct fluorescence was observed with a macroscope 2 weeks later (scale bars, 2 mm [left] and 100 μm [right]). Representative view (Z-stack projection, two experiments, eight eyes). (B) AAV-meganuclease (MEGA) induced MEGA expression. Western blot analysis of the stroma 2 weeks after intrastromal injection (one experiment, n = 4). (C, D) Observation and quantification of stromal X-Gal-generated chromophore (X-Gal-c) release 1 and 6 days postinjection of 25 μL of rHSV-1 solution (2,000 PFUs). Macroscopic examination (C, left panel; scale bar, 2 mm), Z-stack maximum intensity projection of stromal X-Gal-c release (C, central panel; day 1, three experiments with n = 4 eyes; 6 days, two experiments with n = 6 eyes; scale bar, 1 mm), and ImageJ-treated outlines of X-Gal-c in the former (C, right panel). (D) X-Gal-c-colored keratocyte counts, labeled areas and integrated densities on days 1 and 6. AAV-CTRL and AAV-MEGA results are expressed as mean ± SD (day 1, n = 8 vector injection sites; day 6, n = 12 sites). Two-sided Mann-Whitney test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (E) Slit-lamp examinations of the corneas 6 days postinfection. Corneal infiltrates seen in the AAV-CTRL-treated eyes as hazy grayish areas, are not detected in the AAV-MEGA-treated corneas. Narrow slit-beam image (left panel, scale bar, 2 mm) and magnified images (middle and right panels). (F) Epithelial infection 6 days after stromal rHSV-1 injection. Quantification of epithelial X-Gal-c. Macroscopic observation (left panel; scale bar, 2 mm), ImageJ outlines of epithelial X-Gal-c (central panel), and X-Gal-c area analysis (right panel; one representative experiment, n = 12). AU: arbitrary units.

Article Snippet: After selecting a viral vector adequate for corneal endothelium transduction , experiments involved single-stranded AAVs based on the AAV-2 genome, equipped with an expression cassette of an intervening sequence (produced as previously described ): (1) coding for the EGFP [AAV-EGFP]; (2) coding for a meganuclease directed against either the ICP0 gene (HSV-1m4) or the capsid protein VP5 gene encoding UL19 (HSV-1m2; AAV-MEGA) ; (3) noncoding EGFP sequence in antisense orientation, with three stop codons close to the promoter (AAV-CTRL), driven by the human ECMV promoter, and packaged in an AAV-1 capsid (vectors AAV-2/1) (Vectalys).

Techniques: Injection, Plasmid Preparation, In Vivo, Infection, Expressing, Virus, Fluorescence, Western Blot, Generated, Labeling, MANN-WHITNEY