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 ble...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-06-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253123000677 |
| _version_ | 1797854350493614080 |
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| author | Jeong Hyeon Lee Jeong Pil Han Dong Woo Song Geon Seong Lee Beom Seok Choi MinJeong Kim Yeji Lee Seokjoong Kim Hyukjin Lee Su Cheong Yeom |
| author_facet | Jeong Hyeon Lee Jeong Pil Han Dong Woo Song Geon Seong Lee Beom Seok Choi MinJeong Kim Yeji Lee Seokjoong Kim Hyukjin Lee Su Cheong Yeom |
| author_sort | Jeong Hyeon Lee |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-09T20:04:33Z |
| format | Article |
| id | doaj.art-b2eacd899eaa49578e6f6dbdd78f0144 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-09T20:04:33Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-b2eacd899eaa49578e6f6dbdd78f01442023-04-02T06:12:51ZengElsevierMolecular Therapy: Nucleic Acids2162-25312023-06-0132161172In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vectorJeong Hyeon Lee0Jeong Pil Han1Dong Woo Song2Geon Seong Lee3Beom Seok Choi4MinJeong Kim5Yeji Lee6Seokjoong Kim7Hyukjin Lee8Su Cheong Yeom9Graduate School of International Agricultural Technology and Institute of GreenBio Science and Technology, Seoul National University, 1447 Pyeongchang-Ro, Daewha, Pyeongchang, Kangwon 25354, KoreaGraduate School of International Agricultural Technology and Institute of GreenBio Science and Technology, Seoul National University, 1447 Pyeongchang-Ro, Daewha, Pyeongchang, Kangwon 25354, KoreaAavatar Therapeutics, Misadaero, Hanam, Gyeonggi-do, 12925, KoreaGraduate School of International Agricultural Technology and Institute of GreenBio Science and Technology, Seoul National University, 1447 Pyeongchang-Ro, Daewha, Pyeongchang, Kangwon 25354, KoreaToolgen, Inc., 172 Magokjungang-Ro, Gangseo, Seoul 07788, KoreaDepartment of Biochemistry, Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX, USACollege of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 03760, KoreaToolgen, Inc., 172 Magokjungang-Ro, Gangseo, Seoul 07788, KoreaCollege of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 03760, KoreaGraduate School of International Agricultural Technology and Institute of GreenBio Science and Technology, Seoul National University, 1447 Pyeongchang-Ro, Daewha, Pyeongchang, Kangwon 25354, Korea; WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-ro, Gwanank, Seoul 08826, Korea; Corresponding author: Su Cheong Yeom, DVM, PhD, Professor, Graduate School of International Agricultural Technology, Seoul National University, 1447 Pyeongchang-Ro, Daewha, Pyeongchang, Gangwon 25354, Korea.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.http://www.sciencedirect.com/science/article/pii/S2162253123000677MT: Oligonucleotides: Therapies and ApplicationsantithrombinCRISPR-Cas9hemophilia Bin vivo knockinlipid nanoparticle |
| spellingShingle | Jeong Hyeon Lee Jeong Pil Han Dong Woo Song Geon Seong Lee Beom Seok Choi MinJeong Kim Yeji Lee Seokjoong Kim Hyukjin Lee Su Cheong Yeom In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector Molecular Therapy: Nucleic Acids MT: Oligonucleotides: Therapies and Applications antithrombin CRISPR-Cas9 hemophilia B in vivo knockin lipid nanoparticle |
| title | In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector |
| title_full | In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector |
| title_fullStr | In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector |
| title_full_unstemmed | In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector |
| title_short | In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector |
| title_sort | in vivo genome editing for hemophilia b therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non viral vector |
| topic | MT: Oligonucleotides: Therapies and Applications antithrombin CRISPR-Cas9 hemophilia B in vivo knockin lipid nanoparticle |
| url | http://www.sciencedirect.com/science/article/pii/S2162253123000677 |
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