In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector.

Recent therapeutic strategies for hemophilia include long-term therapeutic gene expression using adeno-associated virus (AAV) and rebalancing therapy via the downregulation of anticoagulant pathways. However, these approaches have limitations in immune responses or insufficiency to control acute bleeding. Thus, we developed a therapeutic strategy for hemophilia B by a combined rebalancing and human factor 9 (hF9) gene knockin (KI) using a lipid nanoparticle (LNP) and AAV. Antithrombin (AT; Serpin Family C Member 1 [Serpinc1]) was selected as the target anticoagulation pathway for the gene KI. First, the combined use of LNP-clustered regularly interspaced short palindromic repeats (CRISPR) and AAV donor resulted in 20% insertions or deletions (indels) in Serpinc1 and 67% reduction of blood mouse AT concentration. Second, hF9 coding sequences were integrated into approximately 3% of the target locus. hF9 KI yielded approximately 1,000 ng/mL human factor IX (hFIX) and restored coagulation activity to a normal level. LNP-CRISPR injection caused sustained AT downregulation and hFIX production up to 63 weeks. AT inhibition and hFIX protein-production ability could be maintained by the proliferation of genetically edited hepatocytes in the case of partial hepatectomy. The co-administration of AAV and LNP showed no severe side effects except random integrations. Our results demonstrate hemophilia B therapy by a combination of rebalancing and hF9 KI using LNP and AAV.

Genome editing-mediated knock-in of therapeutic genes ameliorates the disease phenotype in a model of hemophilia.

Recently, clinical trials of adeno-associated virus-mediated replacement therapy have suggested long-term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there remain concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock-in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, APOC3, in which a strong promoter facilitated the expression of knocked-in genes in hepatocytes. We also investigated the effects of APOC3 editing using a small Cas9 protein derived from Campylobacter jejuni (CjCas9) in a hemophilic model. We demonstrated that adeno-associated virus-mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3-humanized mice. Moreover, knock-in-driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off-target editing in vitro or in vivo. Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient APOC3-editing platform, providing insights into the development of further long-term therapeutics for diverse monogenic liver diseases.

In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal.

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

GW2 Functions as an E3 Ubiquitin Ligase for Rice Expansin-Like 1.

Seed size is one of the most important traits determining the yield of cereal crops. Many studies have been performed to uncover the mechanism of seed development. However, much remains to be understood, especially at the molecular level, although several genes involved in seed size have been identified. Here, we show that rice Grain Width 2 (GW2), a RING-type E3 ubiquitin ligase, can control seed development by catalyzing the ubiquitination of expansin-like 1 (EXPLA1), a cell wall-loosening protein that increases cell growth. Microscopic examination revealed that a GW2 mutant had a chalky endosperm due to the presence of loosely packed, spherical starch granules, although the grain shape was normal. Yeast two-hybrid and in vitro pull-down assays showed a strong interaction between GW2 and EXPLA1. In vitro ubiquitination analysis demonstrated that EXPLA1 was ubiquitinated by GW2 at lysine 279 (K279). GW2 and EXPLA1 colocalized to the nucleus when expressed simultaneously. These results suggest that GW2 negatively regulates seed size by targeting EXPLA1 for degradation through its E3 ubiquitin ligase activity.

Comparison of vitrectomized with nonvitrectomized eyes after subtenon injection of triamcinolone acetonide to treat diabetic macular edema: Retrospective comparative Analysis of an interventional case series.

PURPOSE: Triamcinolone acetonide (TA) is an alternative option for diabetic macular edema thanks to its cost-benefit ratio and unique delivery route. We performed this study to compare vitrectomized with nonvitrectomized eyes treated with subtenon TA injection for diabetic macular edema. MATERIALS AND METHODS: We retrospectively reviewed the medical records of patients who had undergone subtenon TA injection for diabetic macular edema treatment. The patients were divided into two groups: Vitrectomized and nonvitrectomized. Visual acuity and central subfield macular thickness (CSMT) were analyzed before injection, at 1 and 3 months after injection. RESULTS: Visual acuity in vitrectomized group improved significantly at 1 month (P = 0.002), but this improvement regressed after 3 months. In the nonvitrectomized group, visual acuity did not improve significantly after 1 month, but it did after 3 months (P = 0.019). The CSMT decreased significantly in both groups at 1 and 3 months (P < 0.001). There were no significant differences between the groups at either 1 or 3 months with regard to either visual improvement or change in CSMT. CONCLUSION: Subtenon TA injection could be an alternative treatment option for diabetic macular edema, both in vitrectomized and in nonvitrectomized eyes. TA seems to take effect earlier and decay faster in vitrectomized eyes.

The circadian changes of intraocular pressure and ocular perfusion pressure after tafluprost compared with travoprost in normal tension glaucoma.

PURPOSE: To compare the 24-h changes of intraocular pressure (IOP) and mean ocular perfusion pressure (MOPP) obtained with tafluprost versus travoprost in patients with normal-tension glaucoma (NTG). METHODS: This study is a randomized crossover study of 50 patients newly diagnosed with NTG who received either tafluprost or travoprost given once at 9 PM for 2 months, after which they were crossed over to the other medication for another 2 months. IOP and blood pressure were measured for 24 h before starting the treatment and after finishing the first and second treatment periods. RESULTS: Forty-one patients completed the study. The mean (±standard deviation) 24-h IOP was 16.8±2.0 mmHg at baseline, 14.4±2.2 mmHg on tafluprost, and 13.6±1.8 mmHg on travoprost. Both prostaglandin monotherapies significantly reduced mean 24-h IOP as compared with baseline (P<0.001, P<0.001, respectively), and travoprost demonstrated a lower mean 24-h IOP than tafluprost (P=0.044). Both treatments significantly reduced the IOP from baseline at every point over 24 h. At 3 individual time points, travoprost provided a lower IOP than tafluprost: at 4 PM (13.8±2.7 vs. 14.8±2.6 mmHg, P=0.041), at 6 PM (13.5±2.5 vs. 14.4±2.5 mmHg, P=0.006), and at 8 PM (13.3±2.5 vs. 14.5±2.4 mmHg, P=0.029). Both tafluprost and travoprost significantly increased the 24-h MOPP (P=0.008, P=0.002, respectively), and travoprost demonstrated a greater 24-h MOPP than tafluprost (P=0.027). CONCLUSIONS: Both tafluprost and travoprost were effective in lowering IOP and increasing MOPP throughout 24 h in NTG. However, travoprost reduced IOP greater than tafluprost in the late afternoon and evening.