Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases

Caneva, Alessia De; Porro, Fabiola; Bortolussi, Giulia; Sola, Riccardo; Lisjak, Michela; Barzel, Adi; Giacca, Mauro; Kay, Mark A.; Vlahoviček, Kristian; Zentilin, Lorena; Muro, Andrés F.

doi:10.1172/jci.insight.128863

Cited by 38 publications

(57 citation statements)

References 65 publications

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“…Instead, gene therapy can provide constant serum level of therapeutic proteins with a single administration and can induce immune tolerance to the expressed transgene 41 . In particular, the idea of integrating a therapeutic transgene in a safe and highly transcribed genomic locus has been already described for the albumin gene 5 – 7 and is now under clinical evaluation (NCT03041324, NCT02695160). However, this in vivo approach is hampered by pre-existing liver conditions, pre-existing neutralizing antibodies and cell-mediated immune responses against AAV vectors used to deliver transgenes or nucleases, thus severely limiting the number of potentially treatable patients 8 .…”

Section: Discussionmentioning

confidence: 99%

“…To overcome this issue, a single “safe harbor” or a highly transcribed genomic locus can be exploited to integrate and overexpress different therapeutic transgenes 5 . Previous studies successfully used adeno-associated virus (AAV) for nuclease-mediated targeting of transgenes under the control of the endogenous albumin promoter in liver 6 , 7 . The striking transcriptional activity of this locus achieved therapeutic protein levels in different preclinical models and thus prompted the first in vivo genome editing trial in humans (NCT03041324).…”

Section: Introductionmentioning

confidence: 99%

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Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

et al. 2020

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Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

et al. 2020

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“…Moreover, researchers can manipulate gene expression at precise sites and to precise degrees of expression. Therefore, these SaCas9 systems can probably be used to edit genes at speci c sites to improve tissue traits and treat chronic diseases [41,42]. First, we report the development of an SaCas9 system with a different sgRNA promoter (tRNA pro ~ 72 bp) and SaCas9 promoter (EF1α pro ~ 212 bp) that can be delivered to cells via rAAV.…”

Section: Discussionmentioning

confidence: 99%

A Newly Built rAAV-SaCas9 Genome Editing System Enables Muscle-Directed Gene Editing to Improve Muscular Atrophy

Weng¹,

Li²,

Zhao³

et al. 2020

Preprint

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Background At present, genome editing at specific sites in vivo is affected by many factors, including the choice of vector, the efficiency of editing proteins and the influence of the internal environment. These factors make gene editing ineffective and even have adverse effects. Methods Here, we report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and we inserted the sequence of the green fluorescent protein eGFP into rAAV. The efficiency of rAAV gene editing in vivo and in vitro was analyzed by time point and virus titer. In addition, we used the rAAV9-SaCas9 system to knock out the myostatin gene in the thigh muscles of muscle-atrophic mice. Results We demonstrated that the gene editing elements regulated by the rAAV-SaCas9 system can be expressed. By increasing the amount of rAAV and the reaction time, the editing efficiency of myostatin and the expression level of eGFP protein can be improved in vitro and vivo. Furthermore, We demonstrated that muscle cells were improved by knockout partial myostatin gene in a mouse model of muscular dystrophy. Conclusions The rAAV-SaCas9 system can be expressed in a stable and long-term manner. The system has substantial therapeutic potential in treating muscular atrophy.

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“…Inducing double strand breaks in this locus will stimulate HDR and integration efficacy. In a follow up study, increased correction was established by combining the generide approach with an AAV- saCas9 guided to the albumin locus [ 62 ]. In all studies neonatal mice were used, modelling the use early after birth, when the liver is actively growing and hepatocytes are proliferating.…”

Section: Gene Therapymentioning

confidence: 99%

“…In this study, life-long therapeutic correction of the pathology was established with a treatment shortly after birth. Further, no off-target integrations were seen in predicted sites which underlines the safety profile of this gene modulation system [ 62 ]. This targeted integration strategy seems a feasible option to treat PFIC shortly after birth.…”

Section: Gene Therapymentioning

confidence: 99%

Gene Therapy for Progressive Familial Intrahepatic Cholestasis: Current Progress and Future Prospects

Bosma¹,

Wits²,

Oude-Elferink³

2020

IJMS

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Progressive Familial Intrahepatic Cholestasis (PFIC) are inherited severe liver disorders presenting early in life, with high serum bile salt and bilirubin levels. Six types have been reported, two of these are caused by deficiency of an ABC transporter; ABCB11 (bile salt export pump) in type 2; ABCB4 (phosphatidylcholine floppase) in type 3. In addition, ABCB11 function is affected in 3 other types of PFIC. A lack of effective treatment makes a liver transplantation necessary in most patients. In view of long-term adverse effects, for instance due to life-long immune suppression needed to prevent organ rejection, gene therapy could be a preferable approach, as supported by proof of concept in animal models for PFIC3. This review discusses the feasibility of gene therapy as an alternative for liver transplantation for all forms of PFIC based on their pathological mechanism. Conclusion: Using presently available gene therapy vectors, major hurdles need to be overcome to make gene therapy for all types of PFIC a reality.

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Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases

Cited by 38 publications

References 65 publications

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

A Newly Built rAAV-SaCas9 Genome Editing System Enables Muscle-Directed Gene Editing to Improve Muscular Atrophy

Gene Therapy for Progressive Familial Intrahepatic Cholestasis: Current Progress and Future Prospects

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